Methods and materials for producing transgenic artiodactyls

ABSTRACT

Swine animal models comprising a genomic disruption of an endogenous gene chosen from the group consisting of a Low-Density Lipoprotein Receptor gene LDLR, Duchene&#39;s Muscular Dystrophy (DMD) gene, and hairless gene (HR). Methods of preparing transfected cells useful for making a transgenic animal comprising exposing a first group of cells to a transfection agent and reseeding the group with additional cells that have not been exposed to the agent. The transgenic animals are useful for medical and scientific animal models of human diseases and conditions, as well as sources for cells, tissues, and biomaterials.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.13/025,373 filed Feb. 11, 2011 which claims priority to U.S. ProvisionalApplication No. 61/303,523 filed Feb. 11, 2010 and 61/309,949 filed Mar.3, 2010 which are hereby incorporated by reference herein.

FIELD OF THE INVENTION

The field of the invention relates to the production of transgenicartiodactyls, for instance pigs. Some aspects of the field relate to thegenes manipulated to make the transgenic animal, for instance the lowdensity lipoprotein receptor (LDLR) gene, Duchene's Muscular Dystrophy(DMD) gene, and the Hairless (HR) gene. Other aspects of the fieldrelate to techniques for transforming swine cells.

BACKGROUND

Swine are an important agricultural commodity and biomedical model.Manipulation of the pig genome provides opportunity to improveproduction efficiency, enhance disease resistance, and add value toswine products. Genetic engineering can also expand the utility of pigsfor modeling human disease, developing clinical treatment methodologies,or donating tissues for xenotransplantation. Heightened interest in suchmodels for human disease and in the production of transgenic livestockfor biomedical applications have increased the need for improved methodsfor transgenesis, as well as for particular models of various diseases.

SUMMARY OF THE INVENTION

Herein is described a method for making a stably transfected swine cell.The arts of transgenic artiodactyl cloning have generally lacked aconsistent and robust technique for making stably transfected swinecells. Once a stably transfected cell is produced, transgenic swine maybe made and then used to meet a growing need for uniform animal modelsof human pathologies.

One aspect of the method is that a first group of cells may be treatedto introduce an exogenous gene and then mixed with a second group ofcells that have not been treated. A series of collection and selectionprocesses may be overlaid with this step being repeated. This method isexemplified herein in the context of the production of swine cells witha knockout for the low density lipoprotein receptor (LDLR), Dystrophingene (DMD), and Hairless gene (HR) in male and female domestic andminiature swine cells.

Transgenic animals with knockouts for LDLR, and DMD gene are describedherein. These animals are useful for modeling atherosclerosis andDuchenne's muscular dystrophy, respectively. Knockouts for HR are usefulfor providing components for medical devices, including dermal derivedbiomaterials and for the use of pigs as models for transdermal drugdelivery, and other applications benefiting from denuded skin.

An embodiment of the invention is a transgenic swine comprising agenomic disruption of an endogenous gene chosen from the groupconsisting of a Low-Density Lipoprotein Receptor gene (LDLR), Duchene'sMuscular Dystrophy (DMD) gene, and hairless gene (HR). Said genomicdisruption may be engineered for preventing expression of a functionalprotein and/or preventing expression on any protein. The swine may behomozygous or heterozygous for said disrupted gene. The swine may befree of a marker gene. The swine may exhibit a phenotype chosen from thegroup consisting of hypercholesterolemia, atherosclerosis, andatherosclerotic lesions. Some of all of the cells in the animal may bedisrupted with respect to the DMD, LDLR or HR gene. The disruption maybe inducible upon administration of an induction agent. The swine may bechosen from the group consisting of pig, miniature pig, and Ossabaw pig.Tissue recovered from such a pig is included, as well as methods ofrecovering said tissue.

An embodiment is a transfected somatic swine cell comprising a disruptedgene chosen from the group consisting of a Low-Density LipoproteinReceptor gene (LDLR), Duchene's Muscular Dystrophy (DMD) gene, and ahairless gene (HR). The cell may be chosen from the group consisting ofembryonic blastomere, fetal fibroblast, adult ear fibroblast, andgranulosa cell. A transgenic swine may be prepared by nuclear transferof such a cell.

An embodiment is a method of introducing an exogenous nucleic acid intoa swine cell in vitro comprising exposing a first group of swine cellsto a transfection agent that comprises an exogenous nucleic acid duringa first culture time period and subsequently adding a second group ofswine cells to the first group for a second culture time period, whereinthe second group of cells have not been exposed to the transfectionagent. The first group of cells may be chosen from the group consistingof primary fetal swine cells and swine fibroblasts. A ratio of thesecond group of cells to the first group of cells may be between 1:1 and20:1. The exogenous nucleic acid may disrupts a target gene chosen fromthe group consisting of a Low-Density Lipoprotein Receptor gene (LDLR),Duchene's Muscular Dystrophy (DMD) gene, and a hairless gene (HR).

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: Depicts the LDLR knockout strategy and PCR results. Panel (A):Schematic of wildtype (wt) and correctly targeted (Targeted) LDLR locusat exon 4 after homologous recombination with the rAAV-LDLR E4-stopreplacement cassette. The rAAV-LDLR E4-stop cassette contains a floxedPGK-Neo selection cassette inserted within exon 4 flanked by a 5′homology arm of 0.9 kb and a 3′ homology arm of 0.3 kb. Panel (B) G-418resistant colonies were screened for gene targeting by amplification ofjunctions between the LDLR locus and the PGK-Neo cassette (indicated asblack triangles in panel (A). Shown is the PCR result from one of the 25μl transduction plates. Positive wells are expected to have a 1.1 Kb PCRproduct whereas negative colonies should have no product. Examples ofstrong (black arrows) and weak signal (light arrows) are shown. Positive(++˜150 copies, +15 copies) and negative (−wild type genomic DNA only)controls are shown in the bottom right corner of the gel.

FIG. 2: Depicts the restriction analysis of the 3′-end of recombinantamplicons in the strategy of FIG. 1. A second set of PCR primers (blueand red triangle) also confirmed accurate targeting at the LDLR locus.PCR amplicons were TOPO-cloned and subjected to restriction analysis andsequencing (data not shown) to confirm homology recombination. Theliberation of a 0.7 Kb band from clones 1, 2, 3, and 5 confirmed theidentity of the recombinant colonies.

FIG. 3: Depicts the results of a Southern analysis of recombinantcolonies. Eight colonies amplified by WGA were subjected to Southernanalysis with the probe indicated in FIG. 1 panel A. The expectedendogenous (E), positive control (+), and targeted band (clones markedwith an asterisk) were observed, although the majority (˜80%) of cellsappear in each clone appear to be wild type.

FIG. 4: Confirmation of round 1 and 3 positives by PCR/Restrictionanalysis. Panel (A) shows a correctly targeted (Targeted) LDLR locus atexon 4 after homologous recombination with the rAAV-LDLR E4 stopcassette. PCR primers for screening 5′ (open triangles) and 3′ (filledtriangles) junctions are shown. Panel (B) depicts 5′ junction PCRperformed on WGA DNA from 7 and 8 colonies identified in the primary PCRscreen of round 1 and 3, respectively. PCR from correctly targetedclones was expected to produce the 1735 bp and its identity is verifiedby restriction digest with EcoRI (labeled “E” in panel (A) resulting in3 fragments (748, 607 and 380 bp) of which the 748 bp band is indicated.WGA DNA from a negative colony was used as the negative control (−).Panel (C) depicts 3′ PCR from correctly targeted colonies that produceda band of 999 bp and with its identity as verified by restriction digestwith XhoI resulting in 2 fragments of 842 (indicated) and 157 bp. Twopositive (++˜150 copies, +15 copies) and two negative controls (WGA DNAfrom a negative colony) are shown. While a band appears in both negativecontrols at approximately 999 bp, it is not cleaved by XhoI indicatingamplification of a random DNA fragment of similar size rather than apositive result. This feature can also be observed in clones 2-5, 9 and12. The PCR was not sensitive enough to detect the 15 copy positive (+)control.

FIG. 5: Confirmation of round 1 and 3 positives by Southern Blotting:Candidates identified by PCR (FIG. 4) were subjected to WGA/Southernblotting. Restriction digest with EcoRI releases a fragment of 2.8 kb inwild type (Wt) cells and a 4.0 kb fragment for correctly targeted(Targeted) cells (see FIG. 1 for schematic). Each colony identifiedconfirmed positive by 5′ and 3′ junction PCR (FIG. 4) displayed signalcharacteristic of a correctly targeted clone. Some variation in signalindicates not all colonies are pure, however, clones 8, 10, 13 and 15appear to contain a majority of heterozygous LDLR knockout cells.

FIG. 6: DMD Exon 7 Replacement. Panel (A) is a schematic that shows bothwild type (Wt) and a correctly targeted (Targeted) DMD locus at exon 7after homologous recombination with the rAAV-DMD E7R replacementcassette. The rAAV-DMD E7R cassette contains a floxed PGK-Neo selectioncassette flanked by approximately 1 kb homology arms upstream anddownstream of exon 7. Homologous recombination between the DMD locus andrAAV-DMD E7R will result in the complete ablation of exon 7 and a frameshift in the full length dystrophin isoform. Panel (B) shows that G-418resistant colonies were screened for gene targeting by amplification ofjunctions between the DMD locus and the PGK-Neo cassette. Both 5′ and 3′junctions could be detected with separate primer pairs (panel A) 5′primers open triangles, 3′ primers filled triangles, allowing forconfirmation of replacement rather than insertion at exon 7. Severalpositive signals are observed for both the 5′ and 3′ ends, often fromidentical wells confirming the presence of correctly targeted cells.Both positive (++˜3,000 copies, +30 copies) and negative (−wild typegenomic DNA only) controls are shown in the bottom right corner of eachgel. Panel (C) shows candidates identified by PCR (panel B) andsubjected to WGA/Southern blotting. Clones correspond to the numberingin panel (B) (clone 1 not shown), and lanes labeled 1′ and 2′ are simplyreplicates created with half volume WGA reactions. Restriction digestwith EcoRI and NcoI (indicated as “E” and “N” in panel A respectively)will release a fragment of 6.2 kb in wild type cells and a 3.3 kbfragment for correctly targeted cells. The strong band observed in forwells 1, 2 and 4 above the 3.3 kb “targeted” band is the predicted sizeof head to tail concatemers of the rAAV-DMD E7R construct. Each wellcontains at some signal at 3.3 kb while wells 3 and 5 contain mostlytargeted cells.

FIG. 7: Confirmation of round 2 positives by PCR/Restriction analysis:The panel (A) schematic shows a correctly targeted (Targeted) dystophinlocus at exon 7 after homologous recombination with the rAAV-DMD E7Rreplacement cassette. PCR primers for screening 5′ (open triangles) and3′ (filled triangles) junctions are shown. Panel (B) shows a 5′ junctionPCR that was performed on WGA DNA from 11 colonies identified in theprimary PCR screen. PCR from correctly targeted clones will produce theexpected 1201 bp and its identity is verified by restriction digest withSpeI (labeled “S” in panel A) resulting in 3 fragments of which the 947bp band is indicated. Eleven male colonies (7-16) and one female colony(17) are shown. (C) 3′ PCR from correctly targeted colonies produces aband of 1165 bp and its identity is verified by restriction digest withHindIII resulting in 2 fragments of 813 and 352 bp. Eleven male colonies(7-16) and one female colony (17) are shown.

FIG. 8: Confirmation of round 2 positives by Southern Blotting. Panel(A) is a schematic of gene targeting at exon 7 of the DYSTROPHIN locus.Panel (B) shows results for candidates identified by PCR (FIG. 7) andsubjected to WGA/Southern blotting. Eleven male colonies (7-16) and onefemale colony (17) are shown. Restriction digest with EcoRI and NcoI(indicated as “E” and “N” in panel A respectively) will release afragment of 6.2 kb in wild type cells and a 3.3 kb fragment forcorrectly targeted cells. Each colony, with the exception of 8 and 11(indicated with arrows) gave positive signal for both 5′ and 3′junctions.

FIG. 9: The porcine Hairless gene (HR) and knockout strategy. (A) Thewild type (Wt) HR gene is comprised of 18 exons, and is located onchromosome 14. The area surrounding exon 2 is highlighted and enlarged.A premature stop codon (TGA) was introduced into exon 2 byrAAV-Homologous recombination to ablate full length HR protein in pigsby truncation of the protein. The pAAV-HR^(TGA) vector includes themajority of exon 2 and homology arms both up and downstream of exon 2.For selection of targeted cells, two version of the HR^(TGA) have beenconstructed, one with a neomycin (Neo) resistance cassette, another witha puromycin (Puro) resistance cassette. Panel (B): This schematic showsthe structure of the targeted HR^(TGA) allele. The HR^(TGA) allele willinterfere with full length HR production in two ways; 1) translationwill be terminated at the engineered TGA stop codon 2) skipping of exon3 by alternative splicing between exons 1 and 3 will cause a frame shiftmutation.

FIG. 10: LDLR Partial CDS from MARC library est sequenced by Applicant.(SEQ ID NO:1).

FIG. 11A sets forth the LDLR HinDIII Subclone sequence (includes exons2-5). (SEQ ID NO:2);

FIG. 11B is a continuation of the sequence of FIG. 11A;

FIG. 11C is a continuation of the sequence of FIGS. 11A and 11B.

DETAILED DESCRIPTION

One embodiment of the invention is a method of transfecting anartiodactyl cell. A first group of artiodactyl cells may be treated tointroduce an exogenous gene and then mixed with a second group ofartiodactyl cells that have not been so treated. This process has beenobserved to produce significant efficiencies and reproducibility. Aseries of working examples are set forth below, followed by a moredetailed overview of this embodiment.

The working examples are also embodiments of the invention. Theseexamples describe stably transfected swine cells made with varioustransformations. These cells may be used to make transgenic animals,which are useful for many purposes including animal models of humandiseases and conditions and sources of tissue.

Hypercholesterolemia Swine Model: Transgenic Pigs with LDLR GeneModification

Cardiovascular disease is a leading cause of death and dysfunction inthe United States, with coronary artery disease being a majorcontributor. Animal models are fundamental to understanding themechanisms of atherosclerosis. The development of new therapies reliesheavily on the use of these models. Unfortunately, there is a lack ofsuitable large-animal models for studying new therapies or testing them.For instance, several stent-drug combinations have been successful inanimal studies but failed in subsequent human clinical trials. Trialsthat are successful in the animal models that subsequently fail in humantrials may be explained by unfaithful replication of human diseasepathology in the animal models.

Hypercholesterolemia is a principal cause of atherosclerosis. Rabbits,swine and rhesus monkeys with genetic mutations linked tohypercholesterolemia have been used to study atherosclerosis and recentresearch has focused on genetically modified mice. However, geneticallymodified mice that manifest hypercholesterolemia do not exhibit lesionstypical of atherosclerosis in humans. Some inbred swine with defectiveLow-Density Lipoprotein Receptors (LDLR) do develop lesions but do notshow a consistent and predictable manifestation of the disease. TheLow-Density Lipoprotein Receptor (LDLR) is a cell surface receptor thatmediates the endocytosis of cholesterol-rich low density lipoprotein(LDL). In humans, the LDLR protein is encoded by the LDLR gene and wasimplicated as having a role in familial hypercholesterolemia (Brown M S,Goldstein J L (1984). “How LDL Receptors Influence Cholesterol andAtherosclerosis”. Scientific American 251: 52-60.).

Herein, as described below, transgenic swine cells were made with adefect in LDLR expression. Transgenic animals may be made from thesecells using any of a variety of standard technique known to artisans inthese fields. In brief, the production of a functional LDLR gene productwas disrupted by introduction of a stop cassette within LDLR exon 4 byAdeno-associated virus (rAAV) homologous recombination (HR). An rAAV HRcassette (rAAV-LDLR-E4-stop) was generated with a PGK-Neo selectioncassette inserted within LDLR exon 4 at the XhoI restriction site (FIG.1). Replacement of LDLR exon 4 with rAAV-LDLR-E4-stop resulted in atruncated, non-functional LDLR protein product. Several AAV constructswere designed and created that either targeted other exons or thatavoided certain repetitive elements around exon 4. Colonies with LDLRnonexpression were identified from three separate transductions.

First Transduction Results

Two million male primary fetal fibroblasts (PFF) were plated in eachwell of a 6-well plate 24 hours prior to incubation (completelyconfluent) with 5, 25, 100, and 150 μl (per well) of rAAV-LDLR-E4-stopviral supernatant in 1 ml of growth medium. Cells were incubated for twohours prior to the addition of 3 ml growth medium. Twenty-four hourslater, cells from each transduction were plated onto five 96-well platesat the density of 2000 cells/well. Cells were allowed to recoverovernight prior to selection in medium containing 250 μg/ml G418,subsequently increased to 300 μg/ml on day 8. On day 16, all the wellswere 100% confluent from the 100 an 150 μl transductions, most wellswere completely confluent from the 25 μl transduction, and approximatelyhalf of the wells from the 5 μl transduction were confluent. On day 17,cells were trypsinized from both 5 and 25 μl transduction plates with 25μl of TRYPLE EXPRESS (Invitrogen, CA) for 5 min under 37 degrees Celsiusafter Phosphate Buffer Solution (PBS) washing. 175 μl of serumcontaining media were added and mixed. Half of the cell suspension wascryopreserved in deep-well plates while the remaining half wastransferred to 96-well PCR plates, pelleted and resuspended in 20 μl of1×PCR lysis buffer.

Second Transduction Results

The second transduction was performed as with the first transductionwith a few exceptions, as follows. PFF cells were plated on 6-wellplates 24 hours prior to transduction to achieve a density of 30%confluence. Twenty-five microliters of rAAV-LDLR E4-stop viralsupernatant was added to 1 ml growth medium and were plated onto five96-well plates at the density of 1,000 cells/well 24 hours later. Cellswere allowed to recover overnight prior to selection in mediumcontaining 300 ug/ml G418. On day 15, most wells were 100% confluent andcells were split and plated on replicate 96-well plate wells. When wellswere 100% confluent, cells from one replicate were collected in 1×PCRlysis buffer.

PCR Analysis

PCR analysis was conducted on the 5 μl and 25 μl transduction platesfrom the first transduction and the 25 μl transduction plates from thesecond transduction using primers designed to amplify across the 3′junction (FIG. 1 Panel A). Approximately 50% of wells were confluent inthe first transduction 5 μl transduction plates, therefore, it would beexpected that most wells would harbor one G-418 resistant colony.Unfortunately, no positive wells were detected from the 5 μl platessuggesting that homologous recombination at the LDLR locus had occurredin less than 1 in 250 G-418 resistant colonies.

Cells from the first transduction and second transduction 25 μltransduction plates were screened next. Unlike the previous firsttransduction 5 μl plates, most wells were 100% confluent and wereexpected to contain 2-6 independent G-418 resistant colonies per well.Despite the likelihood of multiple colonies per well, positive signalsfrom 26 wells of the first transduction and 15 wells of the secondtransduction (FIG. 1 panel B) were detected. Signal intensity of the PCRvaried significantly among positives and likely reflected the proportionof cells that are correctly targeted versus resistant “bystander”colonies containing random integration of the targeting vector. Intotal, 11 of the 41 positives had strong signal and were cryopreservedfor WGA/Southern blot analysis. PCR products from 4 of 5 of the strongpositives were confirmed by restriction digestion (FIG. 2). These cloneswere further analyzed by Southern hybridization (FIG. 3) and identifiedthe expected band for the correctly targeted locus. However, in pureknockout colonies, a 50:50 ratio of intensity between knockout and wildtype alleles were expected, thus clones with the expected knockoutallele appear to be confounded by the presence of wt cells, ˜80% basedon signal intensity (FIG. 3).

Third Transduction for LDLR Disruption

A third infection was conducted in both male and female PFF. Transducedcells were plated at densities of 100/well, 200/well, and 500/well on96-well plates (5 replicates for each density and sex), supplementedwith wild type cells to a total of 1,000 cells per well, and selected inG-418 for two weeks. Neomycin resistant colonies appeared inapproximately 30 to 50 of wells in the 100 and 200 plates while greaterthan 90 percent of wells in 500 plates contained a colony (Table 1).Primary PCR screening was performed on the 100 and 200 plates resultingin 2 and 11 positive wells for male and female cells respectively (Table2). The healthy (1 male and 7 female) colonies were cryopreserved and aportion were set aside for WGA. PCR of both 5′ and 3′ junctions from WGADNA revealed positive signal in 1 and 5 of the male and female coloniesrespectively (FIG. 4 and Table 2). Identity of the junction PCR wasconfirmed by restriction digest (FIG. 4). Finally, positive clones fromboth the first and second transductions were analyzed by Southernblotting confirming the knockout allele in each PCR positive clone (FIG.5).

TABLE 1 96-well plate selection (Third transduction) Plating Wells w/Neo^(R) density Cells (% Colonies (% (cells/well) Wells wells) selected)100 Male 480 188 (39) 188 (0.39) 200 Male 480 222 (46) 222 (0.23) 100Female 480 150 (31) 150 (0.31) 200 Female 480 183 (38) 183 (0.19)

TABLE 2 Targeting Frequency (Third transduction) Neo^(R) 1° PCR 5′-3′PCR RE Frequency Colonies Positives Positives (HR (% selected) (%colonies) (% colonies) positive/selected) Male 20 410 (0.28) 2 (0.48) 1(0.24) 6.94 × 10⁻⁶ Female 17 333 (0.23) 9 (2.7)  5 (3.78) 3.47 × 10⁻⁵

These tests verified that cells were made with LDLR knockouts.Specifically, the following clones contained cells with heterozygousknockout of the LDLR locus; M: clone 1 & F: clones 8, 10, 11, 13, 14,and 15. In addition, the male clones 1 and 7 may contain the knockout,but failed to be verified by WGA: Southern analysis. The cells may becloned into male and female pigs by Somatic Cell nuclear transfer,Chromatin transfer or other suitable techniques. These founders may thenbe bred to create pigs homozygous for knockout of the LDLR gene.

These techniques may be used to produce animals that are homozygous orheterozygous for the disrupted gene; cells that have a marker gene orare free of a marker gene, a swine that exhibits a phenotype chosen fromthe group consisting of hypercholesterolemia, atherosclerosis, andatherosclerotic lesions (including any combination thereof), wherein thedisrupted LDLR gene is disrupted at exon 4, and wherein all of the LDLRgenes in the swine are disrupted.

For this gene and others, techniques for making marker-free recombinantcells and animals has been described in U.S. Publication No.2010/0146655, which is hereby incorporated herein by reference for allpurposes; in case of conflict, the specification is controlling.

The working example described these process with respect to disruptionof exon 4 of LDLR. Familial hypercholestolemia via LDLR mutation iscommonly due to a dominant mutation in LDLR at a variety of locationsranging from exon 1 to deletion in the final three exons (as is known,see LOW DENSITY LIPOPROTEIN RECEPTOR; LDLR-OMIM, hereby incorporated byreference herein). Therefore, frame-shift mutation, truncation, orintroduction of single amino acid changes throughout the LDRL gene areexpected to disrupt LDLR function. Targeting such changes would simplyrequire the acquisition of the pig LDLR sequences available in Genbank,ENSEMBL, or as described in SEQ ID NO:1, and the application of methodsfor homologous recombination, allele conversion, or the introduction ofan indel using zinc finger nucleases, meganucleases, or TAL effectornucleases or any other targeted method for DNA breakage/modification.Like humans, pigs have been described with dyslipidemia on the basis ofmutations in components of the lipid scavenging system. For example, allpig breeds examined are monomorphic at positions in the apolipoprotein E(ApoE) gene that are associated with a predisposition for high plasmaLDL-cholesterol in patients, i.e., they encode arginine residues atpositions 112 and 158 that correspond to the deleterious ApoE4 isoform.Rapacz identified “naturally” occurring hypercholesterolemia in farmpigs leading to atherosclerotic plaques in aged pigs. The causativemutations were identified in two familial hypercholesterolemic pig linesincluding an alternative apolipoprotein B (ApoB) allele and a missensemutation in exon 4 of the low density lipoprotein receptor (LDLR)(R84C). Mutations at the analogous residue in human LDLR (R115C andR115H) have been reported, the latter displaying 64% LDL clearanceactivity compared to wild type LDLR in an in-vitro study. This suggeststhat the R84C mutation in pigs is likely to be a hypomorphic allele ofLDLR. As in humans, pig are dependent on LDLR-mediated removal of LDLfrom circulation since they do not produce ApoB-48 in their livers toallow for ApoE dependent removal of LDL via the chylomicron remnantreceptors, as is the case for rodents and dogs. Ossabaw pigs are ananimal model of hypercholesterolemia; while useful, genetic engineeringof LDDR defects in this breed and others will be have a number ofadvantages for improving these models. One advantage is a severe andrapid onset of dyslipidemia, considering the conservation of LDLR inpigs and humans, as well as the predicted predisposition to high plasmaLDL-cholesterol. The motifs known for truncating, ablating, or otherwisedisrupting LDDR in swine, humans, and mice may accordingly be applied inthe creation of a transfected cell and a transgenic swine.

SEQ ID NO:1 was used to generate a probe for BAC library screening andrecovery of a genomic clone containing the pig LDLR gene. SEQ ID NO:2 isa HinD III subclone from the LDLR BAC that encompasses exons 2-5. Onceavailable, this sequence was verified by comparison to GenBank:FP102365.2.

Further disclosure relating to lipoprotein receptors is provided in U.S.Pat. Nos. 5,521,071, 5,798,209, 6,174,527, 6,833,240, 7,008,776,7,306,794, 7,416,849 and U.S. Publication No. 2002/0155446 which arehereby incorporated herein by reference for all purposes; in case ofconflict, the instant specification is controlling.

Muscular Dystrophy Swine Model: Transgenic Pigs with Dystrophin GeneModification

The primary product of the Dystrophin gene in muscle is dystrophin, a427 kDa rod-shaped protein having four domains: an N-terminal actinbinding domain, 24 triple helix spectrin-like repeats with four hingeregions, a cysteine-rich domain with two potential calcium bindingmotifs, and a unique C-terminal domain (Koenig et al.). In muscle,dystrophin forms a linkage between the cytoskeletal actin and a group ofmembrane proteins, as well as with a number of non-membranal proteins(collectively called dystrophin associated proteins; DAPs) (Yoshida etal., Ervasti et al.). The N-terminal domain binds to the cytoskeletalactin and the association with the DAPs is mediated mainly by thecysteine-rich and C-terminal domains of dystrophin (Suzuki et al., Junget al.). One of the DAPs, α-dystroglycan, binds laminin. Thus, inmuscle, this complex links the cytoskeleton, the sarcolemma and theextracellular matrix (Ahn et al., Campbell, Ozawa et al.).

The Dystrophin gene also codes for two non-muscle isoforms ofdystrophin, each controlled by a different promoter located in the 5′end region of the gene; the brain type dystrophin (Nudel et al., Barneaet al., Boyce et al.) and Purkinje cell type dystrophin (Gorecki etal.). In addition, internal promoters located within downstream intronsfor the dystrophin gene regulate the expression of smaller products.Dp71, a 70.8 kDa protein, consists of only the cysteine-rich andC-terminal domains of dystrophin (Bar et al., Lederfein et al.). It isthe most abundant non-muscle product of the dystrophin gene and has beenfound in all tissues tested so far except for differentiated skeletalmuscle. The highest levels of Dp71 are found in the brain (Rapaport etal., Greenberg et al.). The other known small products of the dystrophingene consist of the cysteine-rich and C-terminal domains with variousextensions into the spectrin-like repeats domain. These products are:Dp116 (Byers et al.), Dp140 (Lidov et al.), and Dp260 (D'Souza et al.),which are expressed mainly in Schwann cells, brain, and retina,respectively, and have molecular weights of 116, 140 and 260 kDa. Thefunctions of the non-muscle dystrophins and of the smaller products ofthe dystrophin gene are not known.

Rodent models of dystrophin have proven invaluable in defining thecomplexity of muscle disease, and enabled the development of severalpromising therapeutic strategies for DMD. However, muscle degenerationin the mdx mouse model is mild in comparison to DMD patients. Forinstance, mdx mice are mobile, they do not have significant fibrosis orjoint contractures, and the skeletal myofibers are only partiallyreplaced by adipose cells later in life. The myotendinous junctions areseverely impaired in DMD patients (Bell, C. D. and Conen, Hasegawa etal., Nagao et al.), but only have minor alterations in maturation andmaintenance in mdx mice (Law and Tidbal). In addition, the loss ofsynaptic folds in the neuromuscular synapse has little effect onsynaptic transmission in mdx mice (Banks et al., Carlson et al., Lyonset al.), but has a greater effect in DMD patients (Slater). Furthermore,the lifespan of mdx mice are only moderately shortened (˜20%) so therelevance of different therapeutic strategies is difficult to assess(Chamberlain et al.). Therapeutic strategies may instead benefit fromexamining large animal models of DMD.

One explanation for the mild phenotype of mdx mice is that thefunctional requirement of dystrophin to transmit muscle forces may beminimal given their small and weak stature in comparison to humans.Satellite cells also retain their regenerative potential better in mdxmice than in DMD patients, so may more actively repair damaged tissue.Another possibility is that homologous proteins (such as utrophin) cancompensate more effectively for the absence of dystrophin in mice.Consistent with this hypothesis, two independent laboratories generatedmice lacking both dystrophin and utrophin to generate a more severemodel of DMD (Deconinck et al., Grady et al.). mdx:utrn−/−mice aresmaller than wild-type mice, develop severe kyphosis, and become lessmobile with age (Deconinck et al., Grady et al.) and they develop aninflammatory response in the skeletal musculature (Deconinck et al.,Grady et al. In these double knockout mice, many of the muscle fibersare replaced by fibrotic tissue that contributes to joint contractures(Deconinck et al., Grady et al.). However, clear differences in thesize, stem cell dynamics, and requirement for dystrophin function argueagainst the continued reliance on rodent models.

There are several cxmd dog models of DMD, including the Golden Retriever(GRMD) (Cooper et al.), Rottweiler (Partridge) German Short-HairedPointer (Schatzberg), and cxmdj Beagles in Japan (Shimatsu et al.).These various dogs display similar although variable phenotypes(Polejaeva, Wheeler and Walters). There is a high mortality rate ofearly neonatal GRMD dogs from selective muscle degeneration (Charreau etal., Kuroiwa et al.). For dogs that live through the neonatal period,muscle degeneration is followed by muscle regeneration and a largeinflammatory response (Nguyen et al.). Some of the muscles have highconcentrations of crystalline calcium and hyaline (Cooper et al., Nguyenet al.) and muscle fibers begin to be replaced by fibrotic tissue andadipose cells at approximately 2 months of age (Nguyen et al.). Jointcontractures are prominent by 6 months and mobility is severelyimpaired. The muscles are atrophic, weaker, and more susceptible tocontraction-induced injury (Nguyen et al., Childers et al.). GRMD dogsdevelop cardiomyopathy (Chetboul and Carlos, et al., Chetboul andEscriou et al.) and respiratory distress that can lead to death(Valentine et al., 1991). GRMD dogs display a mosaic expression oftruncated dystrophins with deletions from exons 2-10 and 4-13(Schatzberg et al.), although expression becomes somewhat more uniformwith age (Cooper et al.). These truncated dystrophins lack part of theN-terminal actin binding domain, hinge 1, spectrin repeat 1, and part ofspectrin repeat 2. The N-terminal actin-binding domain of dystrophin isimportant for dystrophin expression and function (Banks et al., Beggs etal., Le et al., Chelley et al., Le et al., Matsumra et al., Muntoni etal., Novakic et al., Prior et al., Takeshima et al., Winnard et al.).

Although these dogs sometimes present a faithful model of DMD, there issignificant phenotypic variability between dogs with the same mutation(Cooper et al., Shimatsu et al. 2003). In human patients and the GRMDmodel, dystrophin expression can be restored when there is a pointmutation in the N-terminal actin binding domain (Schatzberg et al.,Winnard et al.). Although these regions are important for dystrophinexpression and function, the central actin-binding domain can partiallycompensate for deletions in the N-terminal actin-binding domain (Warneret al., Rybakova et al.). Dp260 can mitigate muscle degeneration whenexpression levels are near normal (Warner et al.). Thus, variations inexpression of the truncated dystrophins in the dog model could explainthe variability in phenotype between dogs. Despite being the only largeanimal model of DMD, the use of dogs is expensive; they are notsusceptible to genetic manipulation, and are not a preferred system dueto the fact that they are an emotive species.

Herein are described cells for making genetically engineered pigs toprovide a superior large animal model of DMD. The size, musculoskeletaland heart physiology of pigs is remarkably similar to humans. The latterfact underlies the widespread use of pigs in cardiovascular research.The size of the pig is will elicit mechanical strain sufficient toinduce DMD in the context of dystrophin deficiency. In addition, thehigh reproductive rate (averaging 10 piglets per litter), and genomemalleability due to cloning put pigs as a large animal model on par withrodents but with greater anatomical and physiological similarity tohumans. Use of gene knockout technology as described herein provides amore robust model.

This method is described in detail herein in the context of theproduction of swine cells with a knockout for the dystrophin gene inmale and female domestic and miniature swine cells. These cells may beused in nuclear transfer to produce DMD−/+founder animals that are bredand expanded through breeding and then used to meet a growing need ofmedical device and pharmaceutical companies for uniform animal models ofhuman pathologies that can help predict the outcome of human therapeuticinterventions.

Establishment of pigs ablated at the dystrophin locus has beenundertaken in both male and female cells. Cloning male cells into pigsby somatic cell nuclear transfer may be used to generate founders thathave DMD, permitting rapid evaluation of the suitability of pigs as amodel. Propagation of pigs ablated at the dystrophin locus may beundertaken using females given its location on the X chromosome. Sows asviable founders will maintain one normal copy of dystrophin and wouldthen need to be bred to generate males with DMD. The results hereinshowed a successful process for knocking out the dystrophin gene in maleand female fibroblasts. These cells are a suitable resource for Somaticcell nuclear transfer or chromatin transfer and will be used to createfounders.

The porcine dystrophin gene was disrupted by recombinantAdeno-associated virus (rAAV) homologous recombination to produce amodel of muscular dystrophy in swine. Homologous recombination betweenthe rAAV cassette and the dystrophin gene would result in thereplacement of exon 7 with a PGK-Neo selection cassette (FIG. 6). Theabsence of exon 7 creates a frame shift in the full length dystrophintranscript eliminating the production of the Dp427 dystrophin isoform.

The experimental approach involved creation of a rAAV replacementcassette (rAAV-DMD E7R) for targeting of DMD exon 7 using a fusion PCRtechnique described in Kohil et al., 2004 (FIG. 6 Panel A). Viralpackaging was conducted by co-transfection of AAV-293 cells withplasmids: rAAV-DMD E7R, pAAV-RC, and pAAV-helper. Two days aftertransfection, cells from one 100 mm plate were lysed in 1 ml of growthmedia by 3× freeze thaw cycles and stored at −80° C. in 300 microliteraliquots.

Viral Transduction Methods:

Early passage pig fetal fibroblasts (PFF) were plated at a density of30,000 cells/cm² in a single well of a six-well plate to achieve 70-80%confluence within 24 hours. Media was changed 1 hour prior totransduction and replaced with 1 ml of fresh growth medium. Onehundred-fifty microliters of viral lysate was added to a single well andincubated under standard growing conditions. After a 24 hour incubation,cells were washed 3× with PBS, trypsinized and plated onto 96 wellplates at densities ranging from 250 cells/well to 2,000 cells/well.Plates seeded at low density were adjusted to 1,000 cells per well withwild type fibroblasts to enhance plating efficiency. On the followingday, medium containing 300 μg/ml G-418 was added and changed 3× in thecourse of two weeks. The surface area in the 96-well plate in this andother experiments was about 0.33 cm² per well, so that a density of 1000cells per well is equal to about 3,000 cells per cm², so that densitiesranged from about 800 to about 6000 cells per cm².

PCR Screen Methods:

After two weeks of selection in G-418, cells were trypsinized anddivided 50:50 between a 96-well PCR plate and a 96-well growth plate.Cells in the PCR plate were pelleted and resuspended in 25 μl of lysisbuffer while the growth plate was returned to the incubator. PCR wasconducted between the PGK-Neo cassette and primers located outside ofboth the 5′ and 3′ homology arms (FIG. 6 Panel B). PCR positive wellswere allowed to grow to confluence prior to trypsinization followed byremoval of cells for Whole Genome Amplification (WGA)/Southern blottingand cryopreservation.

Results; First Transduction:

Early passage PFF were cultured to 70-80% confluence prior totransduction with 150 microliters of viral lysate. Cells weretrypsinized after 24 hours of incubation and plated on 6 wells of 96well plates at densities indicated in Table 3. After 14 days ofselection in G-418, very few viable cells remained in the wild typecontrol wells while each well of the 1,000 and 2,000 cells/well platescontained resistant cells. Most wells in the 500 and 250 plates alsocontained resistant cells (Table 3) however, unlike the 1,000 and 2,000had both partially confluent and empty wells were present. Based on thepercentage of wells containing resistant cells in the 500 and 250plates, we would expect a range of 2-6 and 1-3 independent colonies perwell respectively. These plates were screened for correct targeting byPCR to obtain an estimate of gene targeting frequency for subsequentexperiments. Using PCR primers to amplify both 5′ and 3′ junctions, apositive signal was observed in 6-10% of wells containing resistantcells (FIG. 6 Panel B and Table 4). Wells giving strong signal for both5′ and 3′ junctions were harvested for cryopreservation and WGA/Southernblotting. Since the DMD (dystrophin) gene is on the X chromosome and thecells used for this experiment were male, a pure knockout clone isexpected to completely lack the 6.2 kb wild type allele, while wellscontaining more than one independent resistant colony were likely tocontain both the wild type and 3.3 kb targeted allele. Despite thelikelihood for multiple colonies per well, cells from 2 of the 6 wellsanalyzed by Southern blotting (clones 3 and 5) appeared to containmostly targeted alleles (FIG. 6 Panel B).

TABLE 3 96-well plate selection (First transduction) Plating EmptyPartially density # of wells confluent 100% Confluent (cells/well)plates # (% wells) # (% wells) # (% wells) 250 1 10 (10.4)   54 (56.2)32 (33.3) 500 2 3 (1.8) 120 (71)  45 (27)   1,000 2 0 Nd nd 2,000 1 0 NdNd

TABLE 4 PCR positive wells: (First transduction) Plating density 5′Junction 3′ Junction Both (cells/well) # (% wells^(a)) # (% wells^(a)) #(% wells^(a)) 250  9 (10.5)  9 (10.5)  8 (9.3) 500 12 (7.2) 12 (0.7) 10(6.0) ^(a)Wells containing resistant cells only

Second Transduction:

A second infection was conducted in both male and female PFF. Transducedcells were plated at densities of 100/well, 200/well, and 500/well on96-well plates (5 replicates for each density and sex), supplementedwith wild type cells to a total of 1,000 cells per well, and selected inG-418 for two weeks. Neomycin resistant colonies appeared inapproximately 40, 60, and greater than 90 percent of wells in the 100,200 and 500 plates respectively (Table 5). Primary PCR screening wasperformed on the 100 and 200 plates resulting in 13 and 2 positive wellsfor male and female cells respectively (Table 6). The healthy (11 maleand 1 female) colonies were cryopreserved and a portion was set asidefor WGA. PCR of both 5′ and 3′ junctions from WGA DNA revealed positivesignal in 8 and 1 of the male and female colonies respectively (FIG. 7and Table 6). Identity of the junction PCR was confirmed by restrictiondigest (FIG. 7). Finally, positive clones were analyzed by Southernblotting confirming the knockout allele in each PCR positive clone (FIG.8). The presence of the wt allele in male clones 7, 9, 10, 13 indicatesa mixed population while the wild type allele in the female clone(Serruys et al., 1994) indicates the knockout is heterozygous.

TABLE 5 96-well plate selection (Second transduction) Plating Wells w/NeoR density Cells Colonies (cells/well) Wells (% wells) (% selected)100 Male 480 200 (41) 200 (0.41) 200 Male 480 279 (58) 279 (0.29) 100Female 480 194 (40) 194 (0.40) 200 Female 480 276 (58) 276 (0.29)

TABLE 6 Targeting Frequency (Second transduction) NeoR 1° PCR 5′-3′ PCRRE Frequency Colonies Positives Positives (HR (% selected) (% screened)(% screened) positive/selected) Male 20 479 (0.33) 13 (2.27) 8 (1.67) 5.5 × 10⁻⁵ Female 17 470 (0.33)  2 (0.43) 1 (0.21) 6.94 × 10⁻⁶

These results show a successful knockout of the dystrophin gene in maleand female fibroblasts. These cells are a suitable resource for Somaticcell nuclear transfer and may be used to create founders. The foundersand transgenic wine progeny may have a disrupted DMD gene and exhibit amuscular dystrophy phenotype. The DMD gene may be disrupted, forinstance, at exon 7, or at other sites that are known to disruptproduction of a functional DMD gene product or which have already beenestablished to produce a muscular dystrophy phenotype in other animals.Some or all of the DMD genes may be disrupted. The gene disruption maybe performed to prevent expression of a functional Dp427 dystrophinisoform.

A pig model of DMD may be derived from the introduction of mutantalleles most common amongst DMD patients into the pig dystrophin locusby homologous recombination, which are know, e.g., see DYSTROPHIN;DMD-OMIM, which is hereby incorporated herein by reference for allpurposes; in case of conflict, the specification is controlling (alsoTuffery-Giraud, 2009). Alternatively, ⅓ of the cases of DMD result froma de novo mutation, for which neither parent is a carrier, as is known,see DYSTROPHIN; DMDallelic variants-OMIM, which is hereby incorporatedherein by reference for all purposes; in case of conflict, thespecification is controlling. Common and de novo alleles could bereplicated either by the introduction of previously identified mutantalleles by homologous recombination, gene-conversion, or theintroduction of an indel into relevant exons using zinc finger nuclease,meganucleases, TAL effector nucleases, or any other method for targetedDNA breakage/modification. Artisans are able to access gene sequences asmay be needed, e.g, DMD cDNA Genebank ID: NM_001012408, Genomic withexon 7 NW_001886608 Exon 2-7 Cross referenced with cDNA, Pig Xchromosome NC_010461.2 Cross referenced with cDNA.

Further disclosure relating to DMD genes is provided in U.S. Pat. No.5,239,060 5,430,129, 5,985,846, 6,653,466, and 7,510,867, which arehereby incorporated by reference herein for all purposes; in case ofconflict the instant specification is controlling.

Transgenic Hairless Swine with Disrupted Hairless Gene (HR)

Pig hair is a problematic contaminant for both meat production andderivation of skin derived products. Whereas wild species of swinerequire hair for protection from the sun, hair is not required for thewell-being of modern commercial swine. The hairless (HR) gene encodes anuclear receptor corepressor that is required for hair growth. Humansand rodents lacking a functional HR gene are born with hair, but areunable to regenerate hair follicles resulting in congenital hair lossearly in life (Thompson 2009). Herein, swine cells were made with adisrupted porcine hairless gene (HR) using recombinant Adeno-associatedvirus (rAAV) homologous recombination. The resultant transfected cellsmay be used to produce pigs lacking hair by somatic cell nucleartransfer or chromatin transfer.

An rAAV replacement cassette (pAAV-ssHRTGA) was created for targeting ofswine HR exon 2 using a fusion PCR technique described in Kohil et al.2004. Viral packaging was conducted by co-transfection of AAV-293 cellswith plasmids: pAAV-ssHRTGA, pAAV-RC, and pAAV-helper. Two days aftertransfection, cells from one 100 mm plate were lysed in 1 ml of growthmedia by 3× freeze thaw cycles and stored at −80° C. in 300 microliteraliquots.

Early passage pig fetal fibroblasts were plated at a density of 30,000cells/cm² in a single well of a six-well plate to achieve 70-80%confluence within 24 hours. Media was changed 1 hour prior totransduction and replaced with 1 ml of fresh growth medium. Onehundred-fifty microliters of viral lysate was added to a single well andincubated under standard growing conditions. After a 24 hour incubation,cells were washed 3× with PBS, trypsinized and plated onto 96 wellplates at densities ranging from 125 cells/well to 2,000 cells/well.Plates seeded at low density were adjusted to 1,000 cells per well withwild type fibroblasts to enhance plating efficiency. On the followingday, medium containing 300 μg/ml G-418 was added and changed 3× in thecourse of two weeks. Resistant colonies emerged and were subjected toPCR and Southern analysis. The data indicates a successful knockout ofthe pig ssHR gene in about 15 cell clones to date.

FIG. 9 depicts the porcine Hairless gene (ssHR) and knockout strategy.Panel (A) depicts the wild type (Wt) ssHR gene, which is comprised of 18exons, and is located on chromosome 14. The area surrounding exon 2 ishighlighted and enlarged. A premature stop codon (TGA) was introducedinto exon 2 by rAAV-Homologous recombination to ablate full length ssHRprotein in pigs by truncation of the protein. The pAAV-ssHR^(TGA) vectorincludes the majority of exon 2 and homology arms both up and downstreamof exon 2. For selection of targeted cells, two versions of thessHR^(TGA) were constructed, one with a neomycin (Neo) resistancecassette, another with a puromycin (Puro) resistance cassette. Panel (B)is a schematic that shows the structure of the targeted ssHR^(TGA)allele. The ssHR^(TGA) allele may be used to interfere with full lengthssHR production in two ways; by (1) translation terminated at theengineered TGA stop codon and by (2) skipping of exon 3 by alternativesplicing between exons 1 and 3 to cause a frame shift mutation.

Embodiments include a swine wherein the disrupted endogenous gene isssHR. The swine may exhibit a phenotype chosen from the group consistingof hairlessness and reduced hair (one or both). The swine HR gene may bedisrupted at ssHR exon 2. The HR gene may also be mutated at otherlocations as in humans (artisans are able to identify suitable sequencesas at, e.g., HAIRLESS, MOUSE, HOMOLOG OF; HR-OMIM, which is herebyincorporated herein by reference for all purposes; in case of conflict,the specification is controlling) by homologous recombination, ZFN,TALENs, to create point mutations, frame shift mutation or earlytermination resulting in varying levels of severity. Artisans are ableto access sequences as needed to establish strategies, and guidance forsuch strategies has further been set forth herein; see for instance,Genomic sequence Genebank: NC_010456.2, nucleotides 6341979-6360034Ensembl HR Transcript: NP_001077399.1 (ENSSSCT00000010539).

Further disclosure relating to HR is found in U.S. Publication No.2005/0176665A1 which is hereby incorporated by reference to the extentit does not contradict what is explicitly disclosed herein. Embodimentsof the invention relate to compounds, compositions, and methods for thestudy, diagnosis, and treatment of traits, diseases and conditions thatrespond to the modulation of Hairless (HR) gene expression and/oractivity. Embodiments of the invention are also directed to compounds,compositions, and methods relating to traits, diseases and conditionsthat respond to the modulation of expression and/or activity of genesinvolved in Hairless gene expression pathways or other cellularprocesses that mediate the maintenance or development of such traits,diseases and conditions. Specifically, certain embodiments of theinvention relate to small nucleic acid molecules, such as shortinterfering nucleic acid (siNA), short interfering RNA (siRNA),double-stranded RNA (dsRNA), micro-RNA (mRNA), and short hairpin RNA(shRNA) molecules capable of mediating RNA interference (RNAi) againstHairless gene expression. Such small nucleic acid molecules are useful,for example, in providing compositions to prevent, inhibit, or reducehair growth in a subject, for hair removal or depilation in a subject,or alternately for treatment of traits, diseases and conditions that canrespond to modulation of Hairless gene expression in a subject, such asalopecia and atrichia. In another embodiment, the invention features amethod for treating or preventing a disease, trait, or condition in asubject, comprising administering to the subject a composition of theinvention under conditions suitable for the treatment or prevention ofthe disease, trait or condition in the subject, alone or in conjunctionwith one or more other therapeutic compounds. In yet another embodiment,the invention features a method for inhibiting, reducing or preventinghair growth in a subject comprising administering to the subject acomposition of the invention under conditions suitable for the reductionor prevention of hair growth in the subject. As such, the variousaspects and embodiments are also directed to other genes that areinvolved in Hairless mediated pathways of signal transduction or geneexpression that are involved, for example, in the maintenance and/ordevelopment of hair or hair growth. These additional genes can beanalyzed for target sites using the methods described for Hairless genesherein. Thus, the modulation of other genes and the effects of suchmodulation of the other genes can be performed, determined, and measuredas described herein

Swine Cell Transfection

The transfection of swine cells for generation of transgenic animals iscustomarily a difficult process. As described and demonstrated herein,however, swine cells may be conveniently transfected. A first group ofartiodactyl cells may be transfected and then mixed with a second groupof artiodactyl cells that have not been so treated. Conventionalapproaches rely on treating as many cells as possible to enhance theodds that a cell with a desired genetic trait can be found.Counterintuitively, however, it is better to treat fewer cells and holdback untreated cells to mix with them during subsequent culture. Withoutbeing bound to a theory of operation, the presence of the untreatedcells is believed to produce autocrine and/or paracrine factors thatenhance cell survival or cell phenotype, e.g., activation of morepreferable DNA repair pathways.

A first embodiment of the method involves introducing an exogenousnucleic acid into a swine cell in vitro comprising exposing a firstgroup of swine cells to a vector that comprises an exogenous nucleicacid during a first culture time period and subsequently adding a secondgroup of swine cells to the first group for a second culture timeperiod, wherein the second group of cells have not been exposed to thevector. The first group and the second group, after being combined, arereferred to as a collection, or mixed collection. The mixed collectionmay be subjected to another round of transfection.

One method involves exposing the first group of cells to transfectionagents and then splitting the group into a plurality of cultures. Thecultures of the first cell group may be prepared at various seedingdensities and allowed to grow for a time period and/or until a desiredlevel of confluence is achieved. A second group of cells may be added tothe first group to achieve an overall seeding density and/or afterseeding to achieve a desired cell concentration. This second group maybe cells that have not been exposed to the transfection agents, and maybe wild type cells. The wild type cells may be from the same animal asthe first group of cells, or from a different animal of the same ordifferent species. Any of the groups may also be from a pool of animals,for instance a plurality of swine fetuses. The wild type cells may alsobe from a culture of cells, or a primary or secondary cell culture line.Accordingly, the term wild-type in this specific context of mixing witha group of transfected cells is broad and includes cells transfected byother means. The term native wild-type refers to wild-type cells thathave never been modified.

The ratio of wild-type cells to the first group of cells may be, forexample, between 0.1:1 and 100:1, or between 0.5:1 and 10:1, or betweenabout 1:1 and about 20:1; artisans will immediately appreciate that allthe ranges and values within the explicitly stated ranges arecontemplated.

The first group of cells may be seeded at a first seeding density andwild-type cells co-cultured to achieve a total density or confluence.For instance a group of cells may be exposed to transfection agents andthen seeded into a plurality of cultures at a seeding density (referringto a concentration per area of cells), e.g., from about 100 to about10,000 cells per cm²; artisans will immediately appreciate that all theranges and values within the explicitly stated ranges are contemplated,e.g., about 500 or about 8,000 to about 7,000 or about 10,000. Wild-typecells may then be added to bring the cells to a predeterminedconcentration, e.g., to a value between 1000 and 100,000 cells/cm².

The wild-type cells may be added before, during, or after the seeding ofthe first group of cells. Accordingly, embodiments include seeding thefirst group of cells and the wild type cells within a 24-hour timeperiod, or at essentially the same time. Embodiments also includeseeding the wild-type cells at a time between about 1 day and about 1week before introduction of the first group of cells exposed to thetransfection agents. And embodiments also include seeding the wild-typecells at a time between about 1 day and two weeks after seeding of thefirst group of cells. Artisans will immediately appreciate that all theranges and values within the explicitly stated ranges are contemplated.

These methods may be practiced with the various cell types describedherein, e.g., fibroblasts, primary fetal swine cells, blastomeres. Thecells may be somatic or germ cells. The cells may be an artiodactylcell, e.g., pig, miniature pig, Ossabow pig. The cells may be adult,juvenile, or fetal, and from any of a variety of tissue sources, e.g.,fibroblasts, dermal fibroblasts, dermal, epidermal, mesodermal,mesenchymal, endothelial, vascular, hepatocyte.

These transfection techniques may be used to transfect a cell with anexogenous nucleic acid that disrupts a target gene, e.g., byintroduction of a stop codon or by way of other techniques commonlyavailable to an artisan skilled in the art of preventing expression of anucleic acid in a cell. The genes in the cell may be modified, e.g.,LDLR, DMD, and HR.

Cells transfected as described herein may be used to make transgenicartiodactyls (e.g., pigs, sheep, goats, and cows). The nucleated cellsof the transgenic artiodactyls contain a nucleic acid construct. As usedherein, the term transgenic artiodactyl includes founder transgenicartiodactyls as well as progeny of the founders, progeny of the progeny,and so forth, provided that the progeny retain the nucleic acidconstruct. For example, a transgenic founder animal can be used to breedadditional animals that contain the nucleic acid construct. Transgenicpigs are particularly useful.

Embodiments of the invention include a tissue obtained from thetransgenic artiodactyls (e.g., transgenic pigs) and cells derived fromthe transgenic artiodactyls (e.g., transgenic pigs). As used herein, theterm derived from indicates that the cells can be isolated directly fromthe animal or can be progeny of such cells. For example, an embodimentof the invention is a brain, lung, liver, pancreas, islets, heart andheart valves, muscle, kidney, thyroid, corneal, skin, blood vessel orother connective tissue obtained from a transgenic artiodactyl (e.g.,transgenic pig). Blood and hematopoietic cells, Islets of Langerhans,beta cells, brain cells, hepatocytes, kidney cells, and cells from otherorgans and body fluids, for example, also can be derived from transgenicartiodactyls (e.g., transgenic pigs).

Transgenic Artiodactyls

Various techniques known in the art can be used to introduce nucleicacid constructs into non-human animals to produce founder lines, inwhich the nucleic acid construct is integrated into the genome. Suchtechniques include, without limitation, pronuclear microinjection (U.S.Pat. No. 4,873,191), retrovirus mediated gene transfer into germ lines(Van der Putten et al. (1985) Proc. Natl. Acad. Sci. USA 82, 6148-1652),gene targeting into embryonic stem cells (Thompson et al. (1989) Cell56, 313-321), electroporation of embryos (Lo (1983) Mol. Cell. Biol. 3,1803-1814), sperm mediated gene transfer (Lavitrano et al. (2002) Proc.Natl. Acad. Sci. USA 99, 14230-14235; Lavitrano et al. (2006) Reprod.Fert. Develop. 18, 19-23), and in vitro transformation of somatic cells,such as cumulus or mammary cells, or adult, fetal, or embryonic stemcells, followed by nuclear transplantation (Wilmut et al. (1997) Nature385, 810-813; and Wakayama et al. (1998) Nature 394, 369-374).Pronuclear microinjection, sperm mediated gene transfer, and somaticcell nuclear transfer are particularly useful techniques.

Typically, in pronuclear microinjection, a nucleic acid construct isintroduced into a fertilized egg; 1 or 2 cell fertilized eggs are usedas the pronuclei containing the genetic material from the sperm head andthe egg are visible within the protoplasm. Pronuclear staged fertilizedeggs can be obtained in vitro or in vivo (i.e., surgically recoveredfrom the oviduct of donor animals). In vitro fertilized eggs can beproduced as follows. For example, swine ovaries can be collected at anabattoir, and maintained at 22-28° C. during transport. Ovaries can bewashed and isolated for follicular aspiration, and follicles rangingfrom 4-8 mm can be aspirated into 50 mL conical centrifuge tubes using18 gauge needles and under vacuum. Follicular fluid and aspiratedoocytes can be rinsed through pre-filters with commercial TL-HEPES(Minitube, Verona, Wis.). Oocytes surrounded by a compact cumulus masscan be selected and placed into TCM-199 OOCYTE MATURATION MEDIUM(Minitube, Verona, Wis.) supplemented with 0.1 mg/mL cysteine, 10 ng/mLepidermal growth factor, 10% porcine follicular fluid, 50 μM2-mercaptoethanol, 0.5 mg/ml cAMP, 10 IU/mL each of pregnant mare serumgonadotropin (PMSG) and human chorionic gonadotropin (hCG) forapproximately 22 hours in humidified air at 38.7° C. and 5% CO₂.Subsequently, the oocytes can be moved to fresh TCM-199 maturationmedium which will not contain cAMP, PMSG or hCG and incubated for anadditional 22 hours. Matured oocytes can be stripped of their cumuluscells by vortexing in 0.1% hyaluronidase for 1 minute.

Mature oocytes can be fertilized in 500 μl Minitube PORCPRO IVF MEDIUMSYSTEM (Minitube, Verona, Wis.) in Minitube 5-well fertilization dishes.In preparation for in vitro fertilization (IVF), freshly-collected orfrozen boar semen can be washed and resuspended in PORCPRO IVF Medium to4×10⁵ sperm. Sperm concentrations can be analyzed by computer assistedsemen analysis (SPERMVISION, Minitube, Verona, Wis.). Final in vitroinsemination can be performed in a 10 μl volume at a final concentrationof approximately 40 motile sperm/oocyte, depending on boar. Incubate allfertilizing oocytes at 38.7° C. in 5.0% CO₂ atmosphere for 6 hours. Sixhours post-insemination, presumptive zygotes can be washed twice inNCSU-23 and moved to 0.5 mL of the same medium. This system can produce20-30% blastocysts routinely across most boars with a 10-30% polyspermicinsemination rate.

Linearized nucleic acid constructs can be injected into one of thepronuclei then the injected eggs can be transferred to a recipientfemale (e.g., into the oviducts of a recipient female) and allowed todevelop in the recipient female to produce the transgenic animals. Inparticular, in vitro fertilized embryos can be centrifuged at 15,000×gfor 5 minutes to sediment lipids allowing visualization of thepronucleus. The embryos can be injected with approximately 5 picolitersof the transposon/transposase cocktail using an Eppendorf FEMTOJETinjector and can be cultured until blastocyst formation (˜144 hours) inNCSU 23 medium (see, e.g., PCT Publication No. 2006/036975). Rates ofembryo cleavage and blastocyst formation and quality can be recorded.

Embryos can be surgically transferred into uteri of asynchronousrecipients. For surgical embryo transfer, anesthesia can be induced witha combination of the following: ketamine (2 mg/kg); tiletamine/zolazepam(0.25 mg/kg); xylazine (1 mg/kg); and atropine (0.03 mg/kg) (all fromColumbus Serum). While in dorsal recumbency, the recipients can beaseptically prepared for surgery and a caudal ventral incision can bemade to expose and examine the reproductive tract. Typically, 100-200(e.g., 150-200) embryos can be deposited into the ampulla-isthmusjunction of the oviduct using a 5.5-inch TOMCAT® catheter. Aftersurgery, real-time ultrasound examination of pregnancy can be performedusing an ALOKA 900 ultrasound scanner (Aloka Co. Ltd, Wallingford,Conn.) with an attached 3.5 MHz trans-abdominal probe. Monitoring forpregnancy initiation can begin at 23 days post fusion and can berepeated weekly during pregnancy. Recipient husbandry can be maintainedas normal gestating sows.

In somatic cell nuclear transfer, a transgenic artiodactyl cell (e.g., atransgenic pig cell) such as an embryonic blastomere, fetal fibroblast,adult ear fibroblast, or granulosa cell that includes a nucleic acidconstruct described above, can be introduced into an enucleated oocyteto establish a combined cell. Oocytes can be enucleated by partial zonadissection near the polar body and then pressing out cytoplasm at thedissection area. Typically, an injection pipette with a sharp beveledtip is used to inject the transgenic cell into an enucleated oocytearrested at meiosis 2. In some conventions, oocytes arrested at meiosis2 are termed “eggs.” After producing a porcine embryo (e.g., by fusingand activating the oocyte), the porcine embryo is transferred to theoviducts of a recipient female, about 20 to 24 hours after activation.See, for example, Cibelli et al. (1998) Science 280, 1256-1258 and U.S.Pat. No. 6,548,741. For pigs, recipient females can be checked forpregnancy approximately 20-21 days after transfer of the embryos.

Standard breeding techniques can be used to create animals that arehomozygous for the target nucleic acid from the initial heterozygousfounder animals. Homozygosity may not be required, however. Transgenicpigs described herein can be bred with other pigs of interest.

In some embodiments, a nucleic acid of interest and a selectable markercan be provided on separate transposons and provided to either embryosor cells in unequal amount, where the amount of transposon containingthe selectable marker far exceeds (5-10 fold excess) the transposoncontaining the nucleic acid of interest. Transgenic cells or animalsexpressing the nucleic acid of interest can be isolated based onpresence and expression of the selectable marker. Because thetransposons will integrate into the genome in a precise and unlinked way(independent transposition events), the nucleic acid of interest and theselectable marker are not genetically linked and can easily be separatedby genetic segregation through standard breeding. Thus, transgenicanimals can be produced that are not constrained to retain selectablemarkers in subsequent generations, an issue of some concern from apublic safety perspective.

Once transgenic animal have been generated, expression of a targetnucleic acid can be assessed using standard techniques. Initialscreening can be accomplished by Southern blot analysis to determinewhether or not integration of the construct has taken place. For adescription of Southern analysis, see sections 9.37-9.52 of Sambrook etal., 1989, Molecular Cloning, A Laboratory Manual, second edition, ColdSpring Harbor Press, Plainview; NY. Polymerase chain reaction (PCR)techniques also can be used in the initial screening. PCR refers to aprocedure or technique in which target nucleic acids are amplified.Generally, sequence information from the ends of the region of interestor beyond is employed to design oligonucleotide primers that areidentical or similar in sequence to opposite strands of the template tobe amplified. PCR can be used to amplify specific sequences from DNA aswell as RNA, including sequences from total genomic DNA or totalcellular RNA. Primers typically are 14 to 40 nucleotides in length, butcan range from 10 nucleotides to hundreds of nucleotides in length. PCRis described in, for example PCR Primer: A Laboratory Manual, ed.Dieffenbach and Dveksler, Cold Spring Harbor Laboratory Press, 1995.Nucleic acids also can be amplified by ligase chain reaction, stranddisplacement amplification, self-sustained sequence replication, ornucleic acid sequence-based amplified. See, for example, Lewis (1992)Genetic Engineering News 12, 1; Guatelli et al. (1990) Proc. Natl. Acad.Sci. USA 87, 1874-1878; and Weiss (1991) Science 254, 1292-1293. At theblastocyst stage, embryos can be individually processed for analysis byPCR, Southern hybridization and splinkerette PCR (see, e.g., Dupuy etal. Proc Natl Acad Sci USA (2002) 99(7):4495-4499).

Expression of a nucleic acid sequence encoding a polypeptide in thetissues of transgenic pigs can be assessed using techniques thatinclude, for example, Northern blot analysis of tissue samples obtainedfrom the animal, in situ hybridization analysis, Western analysis,immunoassays such as enzyme-linked immunosorbent assays, andreverse-transcriptase PCR (RT-PCR).

Vectors and Nucleic Acids

A variety of nucleic acids may be introduced into the swine cells, forknockout purposes, or to obtain expression of a gene for other purposes.Nucleic acid constructs that can be used to produce transgenic animalsinclude a target nucleic acid sequence. As used herein, the term nucleicacid includes DNA, RNA, and nucleic acid analogs, and nucleic acids thatare double-stranded or single-stranded (i.e., a sense or an antisensesingle strand). Nucleic acid analogs can be modified at the base moiety,sugar moiety, or phosphate backbone to improve, for example, stability,hybridization, or solubility of the nucleic acid. Modifications at thebase moiety include deoxyuridine for deoxythymidine, and5-methyl-2′-deoxycytidine and 5-bromo-2′-doxycytidine for deoxycytidine.Modifications of the sugar moiety include modification of the 2′hydroxyl of the ribose sugar to form 2′-O-methyl or 2′-O-allyl sugars.The deoxyribose phosphate backbone can be modified to produce morpholinonucleic acids, in which each base moiety is linked to a six membered,morpholino ring, or peptide nucleic acids, in which the deoxyphosphatebackbone is replaced by a pseudopeptide backbone and the four bases areretained. See, Summerton and Weller (1997) Antisense Nucleic Acid DrugDev. 7(3):187-195; and Hyrup et al. (1996) Bioorgan. Med. Chem.4(1):5-23. In addition, the deoxyphosphate backbone can be replacedwith, for example, a phosphorothioate or phosphorodithioate backbone, aphosphoroamidite, or an alkyl phosphotriester backbone.

The target nucleic acid sequence can be operably linked to a regulatoryregion such as a promoter. Regulatory regions can be porcine regulatoryregions or can be from other species. As used herein, operably linkedrefers to positioning of a regulatory region relative to a nucleic acidsequence in such a way as to permit or facilitate transcription of thetarget nucleic acid.

Any type of promoter can be operably linked to a target nucleic acidsequence. Examples of promoters include, without limitation,tissue-specific promoters, constitutive promoters, and promotersresponsive or unresponsive to a particular stimulus. Suitable tissuespecific promoters can result in preferential expression of a nucleicacid transcript in θ cells and include, for example, the human insulinpromoter. Other tissue specific promoters can result in preferentialexpression in, for example, hepatocytes or heart tissue and can includethe albumin or alpha-myosin heavy chain promoters, respectively. Inother embodiments, a promoter that facilitates the expression of anucleic acid molecule without significant tissue- ortemporal-specificity can be used (i.e., a constitutive promoter). Forexample, a beta-actin promoter such as the chicken θ-actin genepromoter, ubiquitin promoter, miniCAGs promoter,glyceraldehyde-3-phosphate dehydrogenase (GAPDH) promoter, or3-phosphoglycerate kinase (PGK) promoter can be used, as well as viralpromoters such as the herpes virus thymidine kinase (TK) promoter, theSV40 promoter, or a cytomegalovirus (CMV) promoter. In some embodiments,a fusion of the chicken θ actin gene promoter and the CMV enhancer isused as a promoter. See, for example, Xu et al. (2001) Hum. Gene Ther.12(5):563-73; and Kiwaki et al. (1996) Hum. Gene Ther. 7(7):821-30.

An example of an inducible promoter is the tetracycline (tet)-onpromoter system, which can be used to regulate transcription of thenucleic acid. In this system, a mutated Tet repressor (TetR) is fused tothe activation domain of herpes simplex VP 16 (transactivator protein)to create a tetracycline-controlled transcriptional activator (tTA),which is regulated by tet or doxycycline (dox). In the absence ofantibiotic, transcription is minimal, while in the presence of tet ordox, transcription is induced. Alternative inducible systems include theecdysone or rapamycin systems. Ecdysone is an insect molting hormonewhose production is controlled by a heterodimer of the ecdysone receptorand the product of the ultraspiracle gene (USP). Expression is inducedby treatment with ecdysone or an analog of ecdysone such as muristeroneA. The agent that is administered to the animal to trigger the induciblesystem is referred to as an induction agent.

Additional regulatory regions that may be useful in nucleic acidconstructs, include, but are not limited to, polyadenylation sequences,translation control sequences (e.g., an internal ribosome entry segment,IRES), enhancers, inducible elements, or introns. Such regulatoryregions may not be necessary, although they may increase expression byaffecting transcription, stability of the mRNA, translationalefficiency, or the like. Such regulatory regions can be included in anucleic acid construct as desired to obtain optimal expression of thenucleic acids in the cell(s). Sufficient expression, however, cansometimes be obtained without such additional elements.

Other elements that can be included on a nucleic acid construct encodesignal peptides or selectable markers. Signal peptides can be used suchthat an encoded polypeptide is directed to a particular cellularlocation (e.g., the cell surface). Non-limiting examples of selectablemarkers include puromycin, adenosine deaminase (ADA), aminoglycosidephosphotransferase (neo, G418, APH), dihydrofolate reductase (DHFR),hygromycin-B-phosphtransferase, thymidine kinase (TK), andxanthin-guanine phosphoribosyltransferase (XGPRT). Such markers areuseful for selecting stable transformants in culture. Other selectablemarkers include fluorescent polypeptides, such as green fluorescentprotein or yellow fluorescent protein.

In some embodiments, a sequence encoding a selectable marker can beflanked by recognition sequences for a recombinase such as, e.g., Cre orFlp. For example, the selectable marker can be flanked by loxPrecognition sites (34 bp recognition sites recognized by the Crerecombinase) or FRT recognition sites such that the selectable markercan be excised from the construct. See, Orban, et al., Proc. Natl. Acad.Sci. (1992) 89 (15): 6861-6865, for a review of Cre/lox technology, andBrand and Dymecki, Dev. Cell (2004) 6(1):7-28. A transposon containing aCre- or Flp-activatable transgene interrupted by a selectable markergene also can be used to obtain transgenic animals with conditionalexpression of a transgene. For example, a promoter driving expression ofthe marker/transgene can be either ubiquitous or tissue-specific, whichwould result in the ubiquitous or tissue-specific expression of themarker in F0 animals (e.g., pigs). Tissue specific activation of thetransgene can be accomplished, for example, by crossing a pig thatubiquitously expresses a marker-interrupted transgene to a pigexpressing Cre or Flp in a tissue-specific manner, or by crossing a pigthat expresses a marker-interrupted transgene in a tissue-specificmanner to a pig that ubiquitously expresses Cre or Flp recombinase.Controlled expression of the transgene or controlled excision of themarker allows expression of the transgene.

In some embodiments, the target nucleic acid encodes a polypeptide. Anucleic acid sequence encoding a polypeptide can include a tag sequencethat encodes a “tag” designed to facilitate subsequent manipulation ofthe encoded polypeptide (e.g., to facilitate localization or detection).Tag sequences can be inserted in the nucleic acid sequence encoding thepolypeptide such that the encoded tag is located at either the carboxylor amino terminus of the polypeptide. Non-limiting examples of encodedtags include glutathione S-transferase (GST) and FLAG™ tag (Kodak, NewHaven, Conn.).

In other embodiments, the target nucleic acid sequence induces RNAinterference against a target nucleic acid such that expression of thetarget nucleic acid is reduced. For example the target nucleic acidsequence can induce RNA interference against a nucleic acid encoding acystic fibrosis transmembrane conductance regulatory (CFTR) polypeptide.For example, double-stranded small interfering RNA (siRNA) or smallhairpin RNA (shRNA) homologous to a CFTR DNA can be used to reduceexpression of that DNA. In one embodiment, the invention features one ormore siNA molecules and methods that independently or in combinationmodulate the expression of Hairless genes encoding proteins, such asproteins comprising Hairless associated with the maintenance and/ordevelopment of hair or hair growth, referred to herein generally asHairless or HR. The description of the various aspects and embodimentsof the invention is provided with reference to exemplary Hairless genereferred to herein as Hairless or HR. However, the various aspects andembodiments are also directed to other Hairless genes, such as Hairlesshomolog genes, transcript variants including HR-1, HR-2 andpolymorphisms (e.g., single nucleotide polymorphism, (SNPs)) associatedwith certain Hairless genes. As such, the various aspects andembodiments are also directed to other genes that are involved inHairless mediated pathways of signal transduction or gene expressionthat are involved, for example, in the maintenance and/or development ofhair or hair growth. Constructs for siRNA can be produced as described,for example, in Fire et al. (1998) Nature 391:806-811; Romano and Masino(1992) Mol. Microbiol. 6:3343-3353; Cogoni et al. (1996) EMBO J.15:3153-3163; Cogoni and Masino (1999) Nature 399:166-169; Misquitta andPaterson (1999) Proc. Natl. Acad. Sci. USA 96:1451-1456; and Kennerdelland Carthew (1998) Cell 95:1017-1026. Constructs for shRNA can beproduced as described by McIntyre and Fanning (2006) BMC Biotechnology6:1. In general, shRNAs are transcribed as a single-stranded RNAmolecule containing complementary regions, which can anneal and formshort hairpins.

Nucleic acid constructs can be methylated using an SssI CpG methylase(New England Biolabs, Ipswich, Mass.). In general, the nucleic acidconstruct can be incubated with S-adenosylmethionine and SssICpG-methylase in buffer at 37° C. Hypermethylation can be confirmed byincubating the construct with one unit of HinP1I endonuclease for 1 hourat 37° C. and assaying by agarose gel electrophoresis.

Nucleic acid constructs can be introduced into embryonic, fetal, oradult porcine cells of any type, including, for example, germ cells suchas an oocyte or an egg, a progenitor cell, an adult or embryonic stemcell, a kidney cell such as a PK-15 cell, an islet cell, a beta cell, aliver cell, or a fibroblast such as a dermal fibroblast, using a varietyof techniques. Non-limiting examples of techniques include the use oftransposon systems, recombinant viruses that can infect cells, orliposomes or other non-viral methods such as electroporation,microinjection, or calcium phosphate precipitation, that are capable ofdelivering nucleic acids to cells.

In transposon systems, the transcriptional unit of a nucleic acidconstruct, i.e., the regulatory region operably linked to a targetnucleic acid sequence, is flanked by an inverted repeat of a transposon.Several transposon systems, including, for example, Sleeping Beauty(see, U.S. Pat. No. 6,613,752 and U.S. Publication No. 2005/0003542);Frog Prince (Miskey et al. (2003) Nucleic Acids Res. 31(23):6873-81);Tol2 (Kawakami (2007) Genome Biology 8(Suppl.1):S7; Minos (Pavlopouloset al. (2007) Genome Biology 8(Suppl.1):S2); Hsmarl (Miskey et al.(2007)) Mol Cell Biol. 27(12):4589-600); and Passport (Leaver (2001)Gene, 271(2), 203-214) have been developed to introduce nucleic acidsinto cells, including mice, human, and pig cells. The Sleeping Beautytransposon is particularly useful. A transposase can be encoded on thesame nucleic acid construct as the target nucleic acid, can beintroduced on a separate nucleic acid construct, or provided as an mRNA(e.g., an in vitro transcribed and capped mRNA).

Insulator elements also can be included in a nucleic acid construct tomaintain expression of the target nucleic acid and to inhibit theunwanted transcription of host genes. See, for example, U.S. PublicationNo. 2004/0203158. Typically, an insulator element flanks each side ofthe transcriptional unit and is internal to the inverted repeat of thetransposon. Non-limiting examples of insulator elements include thematrix attachment region (MAR) type insulator elements and border-typeinsulator elements. See, for example, U.S. Pat. Nos. 6,395,549,5,731,178, 6,100,448, and 5,610,053, and U.S. Publication No.2004/0203158.

Nucleic acids can be incorporated into vectors. A vector is a broad termthat includes any specific DNA segment that is designed to move from acarrier into a target DNA. A vector may be referred to as an expressionvector, or a vector system, which is a set of components needed to bringabout DNA insertion into a genome or other targeted DNA sequence such asan episome, plasmid, or even virus/phage DNA segment. Vector systemssuch as viral vectors (e.g., retroviruses, adeno-associated virus andintegrating phage viruses), and non-viral vectors (e.g., transposons)used for gene delivery in animals have two basic components: 1) a vectorcomprised of DNA (or RNA that is reverse transcribed into a cDNA) and 2)a transposase, recombinase, or other integrase enzyme that recognizesboth the vector and a DNA target sequence and inserts the vector intothe target DNA sequence. Vectors most often contain one or moreexpression cassettes that comprise one or more expression controlsequences, wherein an expression control sequence is a DNA sequence thatcontrols and regulates the transcription and/or translation of anotherDNA sequence or mRNA, respectively.

Many different types of vectors are known. For example, plasmids andviral vectors, e.g., retroviral vectors, are known. Mammalian expressionplasmids typically have an origin of replication, a suitable promoterand optional enhancer, and also any necessary ribosome binding sites, apolyadenylation site, splice donor and acceptor sites, transcriptionaltermination sequences, and 5′ flanking non-transcribed sequences.Examples of vectors include: plasmids (which may also be a carrier ofanother type of vector), adenovirus, adeno-associated virus (AAV),lentivirus (e.g., modified HIV-1, SIV or FIV), retrovirus (e.g., ASV,ALV or MoMLV), and transposons (e.g., Sleeping Beauty, P-elements,Tol-2, Frog Prince, piggyBac).

As used herein, the term nucleic acid refers to both RNA and DNA,including, for example, cDNA, genomic DNA, synthetic (e.g., chemicallysynthesized) DNA, as well as naturally occurring and chemically modifiednucleic acids, e.g., synthetic bases or alternative backbones. A nucleicacid molecule can be double-stranded or single-stranded (i.e., a senseor an antisense single strand). The term transgenic is used broadlyherein and refers to a genetically modified organism or geneticallyengineered organism whose genetic material has been altered usinggenetic engineering techniques. A knockout artiodactyl is thustransgenic regardless of whether or not exogenous genes or nucleic acidsare expressed in the animal or its progeny.

The publications, patents, and patent applications referenced in thisdocument are hereby incorporated by reference herein in their entiretyfor all purposes; in case of conflict, the specification controls.Headings in this document are provided for reference and are notlimiting with respect to the scope of the embodiments of the invention.

REFERENCES

-   Daugherty A. Mouse models of atherosclerosis. Am J Med Sci 2002;    323(1):3-10.-   Rapacz J, Hasler-Rapacz J, Taylor K M, Checovich W J, Attie A D.    Lipoprotein mutations in pigs are associated with elevated plasma    cholesterol and atherosclerosis. Science 1986; 234(4783):1573-7.-   Hasler-Rapacz J, Ellegren H, Fridolfsson A K, et al. Identification    of a mutation in the low density lipoprotein receptor gene    associated with recessive familial hypercholesterolemia in swine. Am    J Med Genet 1998; 76(5):379-86.-   Prescott M F, McBride C H, Hasler-Rapacz J, Von Linden J, Rapacz J.    Development of complex atherosclerotic lesions in pigs with    inherited hyper-LDL cholesterolemia bearing mutant alleles for    apolipoprotein B. Am J Pathol 1991; 139(1):139-47.-   Lee D M, Mok T, Hasler-Rapacz J, Rapacz J. Concentrations and    compositions of plasma lipoprotein subfractions of Lpb5-Lpu1    homozygous and heterozygous swine with hypercholesterolemia. J Lipid    Res 1990; 31(5): 839-47.-   Lowe S W, Checovich W J, Rapacz J, Attie A D. Defective receptor    binding of low density lipoprotein from pigs possessing mutant    apolipoprotein B alleles. J Biol Chem 1988; 263(30):15467-73.-   Grunwald K A, Schueler K, Uelmen P J, et al. Identification of a    novel Arg->Cys mutation in the LDL receptor that contributes to    spontaneous hypercholesterolemia in pigs. J Lipid Res 1999;    40(3):475-85.-   Aiello R J, Nevin D N, Ebert D L, et al. Apolipoprotein B and a    second major gene locus contribute to phenotypic variation of    spontaneous hypercholesterolemia in pigs. Arterioscler Thromb 1994;    14(3):409-19.-   Hirata R, Chamberlain J, Dong R, Russell D W. Targeted transgene    insertion into human chromosomes by adeno-associated virus vectors.    Nat Biotechnol 2002; 20(7):735-8.-   Hirata R K, Xu C, Dong R, Miller D G, Ferguson S, Russell D W.    Efficient PRNP gene targeting in bovine fibroblasts by    adeno-associated virus vectors. Cloning Stem Cells 2004; 6(1):31-6.-   Fattori R, Piva T. Drug-eluting stents in vascular intervention.    Lancet 2003; 361(9353):247-9.-   Holmes D R, Jr., Savage M, LaBlanche J M, et al. Results of    Prevention of REStenosis with Tranilast and its Outcomes (PRESTO)    trial. Circulation 2002; 106(10):1243-50.-   Investigators T E. Acute platelet inhibition with abciximab does not    reduce in-stent restenosis (ERASER study). The ERASER Investigators.    Circulation 1999; 100(8):799-806.-   vom Dahl J, Dietz U, Haager P K, et al. Rotational atherectomy does    not reduce recurrent in-stent restenosis: results of the angioplasty    versus rotational atherectomy for treatment of diffuse in-stent    restenosis trial (ARTIST). Circulation 2002; 105(5):583-8.-   Topol E J, Mark D B, Lincoff A M, et al. Outcomes at 1 year and    economic implications of platelet glycoprotein IIb/IIIa blockade in    patients undergoing coronary stenting: results from a multicentre    randomised trial. EPISTENT Investigators. Evaluation of Platelet    IIb/IIIa Inhibitor for Stenting. Lancet 1999; 354(9195):2019-24.-   Serruys P W, Kay I P, Disco C, Deshpande N V, de Feyter P J.    Periprocedural quantitative coronary angiography after Palmaz-Schatz    stent implantation predicts the restenosis rate at six months:    results of a meta-analysis of the BElgian NEtherlands Stent study    (BENESTENT) I, BENESTENT II Pilot, BENESTENT II and MUSIC trials.    Multicenter Ultrasound Stent In Coronaries. J Am Coll Cardiol 1999;    34(4): 1067-74.-   Serruys P W, de Jaegere P, Kiemeneij F, et al. A comparison of    balloon-expandable-stent implantation with balloon angioplasty in    patients with coronary artery disease. Benestent Study Group. N Engl    J Med 1994; 331(8):489-95.-   Moer R, Myreng Y, Molstad P, et al. Stenting in small coronary    arteries (SISCA) trial. A randomized comparison between balloon    angioplasty and the heparin-coated beStent. J Am Coll Cardiol 2001;    38(6):1598-603.-   Muni N I, Gross T P. Problems with drug-eluting coronary stents—the    FDA perspective. N Engl J Med 2004; 351(16):1593-5.-   Lafont A. The Cypher stent: no longer efficacious at three months in    the porcine model? Cardiovasc Res 2004; 63(4):575-6.-   McFadden E P, Stabile E, Regar E, et al. Late thrombosis in    drug-eluting coronary stents after discontinuation of antiplatelet    therapy. Lancet 2004; 364(9444):1519-21.-   Tumbleson M E, Schook L B. Advances in swine in biomedical research.    New York: Plenum Press, 1996.-   Mahley R W, Weisgraber K H. An electrophoretic method for the    quantitative isolation of human and swine plasma lipoproteins.    Biochemistry 1974; 13(9):1964-9.-   Ruof J, Klein G, Marz W, Wollschlager H, Neiss A, Wehling M.    Lipid-lowering medication for secondary prevention of coronary heart    disease in a German outpatient population: the gap between treatment    guidelines and real life treatment patterns. Prev Med 2002;    35(1):48-53.-   Marz W, Wollschlager H, Klein G, Neiss A, Wehling M. Safety of    low-density lipoprotein cholestrol reduction with atorvastatin    versus simvastatin in a coronary heart disease population (the    TARGET TANGIBLE trial). Am J Cardiol 1999; 84(1):7-13.-   Kuivenhoven J A, Jukema J W, Zwinderman A H, et al. The role of a    common variant of the cholesteryl ester transfer protein gene in the    progression of coronary atherosclerosis. The Regression Growth    Evaluation Statin Study Group. N Engl J Med 1998; 338(2):86-93.-   McPherson R, Hanna K, Agro A, Braeken A. Cerivastatin versus branded    pravastatin in the treatment of primary hypercholesterolemia in    primary care practice in Canada: a one-year, open-label, randomized,    comparative study of efficacy, safety, and cost-effectiveness. Clin    Ther 2001; 23(9): 1492-507.-   Henwood J M, Heel R C. Lovastatin. A preliminary review of its    pharmacodynamic properties and therapeutic use in hyperlipidaemia.    Drugs 1988; 36(4):429-54.-   Harris A. Towards an ovine model of cystic fibrosis. Hum Mol Genet    1997; 6(13):2191-4.-   Coleman R A. Of mouse and man—what is the value of the mouse in    predicting gene expression in humans? Drug Discov Today 2003;    8(6):233-5.-   Marx J. Medicine. Building better mouse models for studying cancer.    Science 2003; 299(5615): 1972-5.-   Hann B, Balmain A. Building ‘validated’ mouse models of human    cancer. Curr Opin Cell Biol 2001; 13(6):778-84.-   Janus C, Westaway D. Transgenic mouse models of Alzheimer's disease.    Physiol Behav 2001; 73(5):873-86.-   Lo D. Animal models of human disease. Transgenic and knockout models    of autoimmunity: Building a better disease? Clin Immunol    Immunopathol 1996; 79(2):96-104.-   Mehlhop P D, van de Rijn M, Goldberg A B, et al. Allergen-induced    bronchial hyperreactivity and eosinophilic inflammation occur in the    absence of IgE in a mouse model of asthma. Proc Natl Acad Sci USA    1997; 94(4): 1344-9.-   Grisham J W. Interspecies comparison of liver carcinogenesis:    implications for cancer risk assessment. Carcinogenesis 1997;    18(1):59-81.-   Piedrahita J A. Targeted modification of the domestic animal genome.    Theriogenology 2000; 53(1):105-16.-   Polejaeva I A. Cloning pigs: advances and applications. Reprod Suppl    2001; 58:293-300.-   Wheeler M B, Walters E M. Transgenic technology and applications in    swine. Theriogenology 2001; 56(8): 1345-69.-   Charreau B, Tesson L, Soulillou J P, Pourcel C, Anegon I.    Transgenesis in rats: technical aspects and models. Transgenic Res    1996; 5(4):223-34.-   Kuroiwa Y, Kasinathan P, Matsushita H, et al. Sequential targeting    of the genes encoding immunoglobulin-mu and prion protein in cattle.    Nat Genet 2004; 36(7):775-80.-   Kolber-Simonds D, Lai L, Watt S R, et al. Production of    alpha-1,3-galactosyltransferase null pigs by means of nuclear    transfer with fibroblasts bearing loss of heterozygosity mutations.    Proc Natl Acad Sci USA 2004; 101(19):7335-40.-   Glass C K, Witztum J L. Atherosclerosis. the road ahead. Cell 2001;    104(4):503-16.-   Bernstein D. Exercise assessment of transgenic models of human    cardiovascular disease. Physiol Genomics 2003; 13(3):217-26.-   Guidance for the Submission of Research and Marketing Application    for Interventional Cardiology Devices: PTCA Catheters, Atherectomy    Catheters, Lasers, Intravascular Stents. Food and Drug    Administration, Center for Devices and Radiological Health, U.S.    Dept. Health and Human Service, 1995.-   Veniant M M, Withycombe S, Young S G. Lipoprotein size and    atherosclerosis susceptibility in Apoe(−/−) and Ldlr(−/−) mice.    Arterioscler Thromb Vasc Biol 2001; 21(10):1567-70.-   Badimon L, Chesebro J H, Badimon J J. Thrombus formation on ruptured    atherosclerotic plaques and rethrombosis on evolving thrombi.    Circulation 1992; 86(6 Suppl):III74-85.-   Grainger D J, Reckless J, McKilligin E. Apolipoprotein E modulates    clearance of apoptotic bodies in vitro and in vivo, resulting in a    systemic proinflammatory state in apolipoprotein E-deficient mice. J    Immunol 2004; 173(10):6366-75.-   Kohila, T., E. Parkkonen, et al. (2004). “Evaluation of the effects    of aluminium, ethanol and their combination on rat brain    synaptosomal integral proteins in vitro and after 90-day oral    exposure.” Arch Toxicol 78(5): 276-282.-   Thompson, C. C. (2009). “Hairless is a nuclear receptor corepressor    essential for skin function.” Nucl Recept Signal 7: e010.-   Rapacz J, Hasler-Rapacz J. Animal Models: The Pig. In: Sparkes R S,    Lusis A J, eds. Genetic factors in atherosclerosis: approaches and    model systems. Basel; New York: Karger, 1989: 139-169.-   Koenig, M., Monaco, A. P. and Kunkel, L. M. (1988) The complete    sequence of dystrophin predicts a rod-shaped cytoskeletal protein.    Cell, 53, 219-228.-   Yoshida, M. and Ozawa, E. (1990) Glycoprotein complex anchoring    dystrophin to sarcolemma. Journal of biochemistry, 108, 748-752.-   Ervasti, J. M., Ohlendieck, K., Kahl, S. D., Gayer, M. G. and    Campbell, K. P. (1990) Deficiency of a glycoprotein component of the    dystrophin complex in dystrophic muscle. Nature, 345, 315-319.-   Suzuki, A., Yoshida, M., Yamamoto, H. and Ozawa, E. (1992)    Glycoprotein-binding site of dystrophin is confined to the    cysteine-rich domain and the first half of the carboxy-terminal    domain. FEBS letters, 308, 154-160.-   Jung, D., Yang, B., Meyer, J., Chamberlain, J. S. and    Campbell, K. P. (1995) Identification and characterization of the    dystrophin anchoring site on beta-dystroglycan. The Journal of    biological chemistry, 270, 27305-27310.-   Ahn, A. H. and Kunkel, L. M. (1993) The structural and functional    diversity of dystrophin. Nature genetics, 3, 283-291.-   Campbell, K. P. (1995) Three muscular dystrophies: loss of    cytoskeleton-extracellular matrix linkage. Cell, 80, 675-679.-   Ozawa, E., Yoshida, M., Suzuki, A., Mizuno, Y., Hagiwara, Y. and    Noguchi, S. (1995) Dystrophin-associated proteins in muscular    dystrophy. Hum Mol Genet, 4 Spec No, 1711-1716.-   Nudel, U., Zuk, D., Einat, P., Zeelon, E., Levy, Z., Neuman, S. and    Yaffe, D. (1989) Duchenne muscular dystrophy gene product is not    identical in muscle and brain. Nature, 337, 76-78.-   Barnea, E., Zuk, D., Simantov, R., Nudel, U. and Yaffe, D. (1990)    Specificity of expression of the muscle and brain dystrophin gene    promoters in muscle and brain cells. Neuron, 5, 881-888.-   Boyce, F. M., Beggs, A. H., Feener, C. and Kunkel, L. M. (1991)    Dystrophin is transcribed in brain from a distant upstream promoter.    Proc Natl Acad Sci USA, 88, 1276-1280.-   Gorecki, D. C., Monaco, A. P., Deny, J. M., Walker, A. P.,    Barnard, E. A. and Barnard, P. J. (1992) Expression of four    alternative dystrophin transcripts in brain regions regulated by    different promoters. Hum Mol Genet, 1, 505-510.-   Bar, S., Barnea, E., Levy, Z., Neuman, S., Yaffe, D. and    Nudel, U. (1990) A novel product of the Duchenne muscular dystrophy    gene which greatly differs from the known isoforms in its structure    and tissue distribution. The Biochemical journal, 272, 557-560.-   Lederfein, D., Levy, Z., Augier, N., Mornet, D., Morris, G., Fuchs,    O., Yaffe, D. and Nudel, U. (1992) A 71-kilodalton protein is a    major product of the Duchenne muscular dystrophy gene in brain and    other nonmuscle tissues. Proc Natl Acad Sci USA, 89, 5346-5350.-   Rapaport, D., Lederfein, D., den Dunnen, J. T., Grootscholten, P.    M., Van Ommen, G. J., Fuchs, O., Nudel, U. and Yaffe, D. (1992)    Characterization and cell type distribution of a novel, major    transcript of the Duchenne muscular dystrophy gene. Differentiation;    research in biological diversity, 49, 187-193.-   Greenberg, D. S., Schatz, Y., Levy, Z., Pizzo, P., Yaffe, D. and    Nudel, U. (1996) Reduced levels of dystrophin associated proteins in    the brains of mice deficient for Dp71. Hum Mol Genet, 5, 1299-1303.-   Byers, T. J., Lidov, H. G. and Kunkel, L. M. (1993) An alternative    dystrophin transcript specific to peripheral nerve. Nature genetics,    4, 77-81.-   Lidov, H. G., Selig, S. and Kunkel, L. M. (1995) Dp140: a novel 140    kDa CNS transcript from the dystrophin locus. Hum Mol Genet, 4,    329-335.-   D'Souza, V. N., Nguyen, T. M., Morris, G. E., Karges, W.,    Pillers, D. A. and Ray, P. N. (1995) A novel dystrophin isoform is    required for normal retinal electrophysiology. Hum Mol Genet, 4,    837-842.-   Cox, G. A., Sunada, Y., Campbell, K. P. and    Chamberlain, J. S. (1994) Dp71 can restore the dystrophin-associated    glycoprotein complex in muscle but fails to prevent dystrophy.    Nature genetics, 8, 333-339.-   Greenberg, D. S., Sunada, Y., Campbell, K. P., Yaffe, D. and    Nudel, U. (1994) Exogenous Dp71 restores the levels of dystrophin    associated proteins but does not alleviate muscle damage in mdx    mice. Nature genetics, 8, 340-344.-   Leibovitz, S., Meshorer, A., Fridman, Y., Wieneke, S., Jockusch, H.,    Yaffe, D. and Nudel, U. (2002) Exogenous Dp71 is a dominant negative    competitor of dystrophin in skeletal muscle. Neuromuscul Disord, 12,    836-844.-   Bell, C. D. and Conen, P. E. (1968) Histopathological changes in    Duchenne muscular dystrophy. J Neurol Sci, 7, 529-544.-   Hasegawa, T., Matsumura, K., Hashimoto, T., Ikehira, H., Fukuda, H.    and Tateno, Y. (1992) [Intramuscular degeneration process in    Duchenne muscular dystrophy—investigation by longitudinal M R    imaging of the skeletal muscles]. Rinsho Shinkeigaku, 32, 333-335.-   Nagao, H., Morimoto, T., Sano, N., Takahashi, M., Nagai, H., Tawa,    R., Yoshimatsu, M., Woo, Y. J. and Matsuda, H. (1991) [Magnetic    resonance imaging of skeletal muscle in patients with Duchenne    muscular dystrophy—serial axial and sagittal section studies]. No To    Hattatsu, 23, 39-43.-   Law, D. J. and Tidball, J. G. (1993) Dystrophin deficiency is    associated with myotendinous junction defects in prenecrotic and    fully regenerated skeletal muscle. Am J Pathol, 142, 1513-1523.-   Banks, G. B., Choy, P. T., Lavidis, N. A. and Noakes, P. G. (2003)    Neuromuscular synapses mediate motor axon branching and motoneuron    survival during the embryonic period of programmed cell death.    Developmental biology, 257, 71-84.-   Carlson, C. G. and Roshek, D. M. (2001) Adult dystrophic (mdx)    endplates exhibit reduced quantal size and enhanced quantal    variation. Pflugers Arch, 442, 369-375.-   Lyons, P. R. and Slater, C. R. (1991) Structure and function of the    neuromuscular junction in young adult mdx mice. J Neurocytol, 20,    969-981.-   Slater, C. R. (2003) Structural determinants of the reliability of    synaptic transmission at the vertebrate neuromuscular junction. J    Neurocytol, 32, 505-522.-   Chamberlain, J. S., Metzger, J., Reyes, M., Townsend, D. and    Faulkner, J. A. (2007) Dystrophin-deficient mdx mice display a    reduced life span and are susceptible to spontaneous    rhabdomyosarcoma. Faseb J, 21, 2195-2204.-   Deconinck, A. E., Rafael, J. A., Skinner, J. A., Brown, S. C.,    Potter, A. C., Metzinger, L., Watt, D. J., Dickson, J. G.,    Tinsley, J. M. and Davies, K. E. (1997)    Utrophin-dystrophin-deficient mice as a model for Duchenne muscular    dystrophy. Cell, 90, 717-727.-   Grady, R. M., Teng, H., Nichol, M. C., Cunningham, J. C.,    Wilkinson, R. S. and Sanes, J. R. (1997) Skeletal and cardiac    myopathies in mice lacking utrophin and dystrophin: a model for    Duchenne muscular dystrophy. Cell, 90, 729-738.-   Cooper, B. J., Winand, N. J., Stedman, H., Valentine, B. A.,    Hoffman, E. P., Kunkel, L. M., Scott, M. O., Fischbeck, K. H.,    Kornegay, J. N., Avery, R. J. et al. (1988) The homologue of the    Duchenne locus is defective in X-linked muscular dystrophy of dogs.    Nature, 334, 154-156.-   Partridge, T. (1997), Models of dystrophinopathy, pathological    mechanisms and assessment of therapies. Cambridge University Press,    Cambridge, pp. 310-311.-   Schatzberg, S. J., Olby, N. J., Breen, M., Anderson, L. V.,    Langford, C. F., Dickens, H. F., Wilton, S. D., Zeiss, C. J.,    Binns, M. M., Kornegay, J. N. et al. (1999) Molecular analysis of a    spontaneous dystrophin ‘knockout’ dog. Neuromuscul Disord, 9,    289-295.-   Shimatsu, Y., Katagiri, K., Furuta, T., Nakura, M., Tanioka, Y.,    Yuasa, K., Tomohiro, M., Kornegay, J. N., Nonaka, I. and    Takeda, S. (2003) Canine X-linked muscular dystrophy in Japan    (CXMDJ). Experimental animals/Japanese Association for Laboratory    Animal Science, 52, 93-97.-   Shimatsu, Y., Yoshimura, M., Yuasa, K., Urasawa, N., Tomohiro, M.,    Nakura, M., Tanigawa, M., Nakamura, A. and Takeda, S. (2005) Major    clinical and histopathological characteristics of canine X-linked    muscular dystrophy in Japan, CXMDJ. Acta Myol, 24, 145-154.-   Valentine, B. A., Winand, N. J., Pradhan, D., Moise, N. S., de    Lahunta, A., Kornegay, J. N. and Cooper, B. J. (1992) Canine    X-linked muscular dystrophy as an animal model of Duchenne muscular    dystrophy: a review. American journal of medical genetics, 42,    352-356.-   Valentine, B. A. and Cooper, B. J. (1991) Canine X-linked muscular    dystrophy: selective involvement of muscles in neonatal dogs.    Neuromuscul Disord, 1, 31-38.-   Valentine, B. A., Cooper, B. J., de Lahunta, A., O'Quinn, R. and    Blue, J. T. (1988) Canine X-linked muscular dystrophy. An animal    model of Duchenne muscular dystrophy: clinical studies. J Neurol    Sci, 88, 69-81.-   Nguyen, F., Cherel, Y., Guigand, L., Goubault-Leroux, I. and    Wyers, M. (2002) Muscle lesions associated with dystrophin    deficiency in neonatal golden retriever puppies. Journal of    comparative pathology, 126, 100-108.-   Childers, M. K., Okamura, C. S., Bogan, D. J., Bogan, J. R.,    Petroski, G. F., McDonald, K. and Kornegay, J. N. (2002) Eccentric    contraction injury in dystrophic canine muscle. Archives of physical    medicine and rehabilitation, 83, 1572-1578.-   Chetboul, V., Carlos, C., Blot, S., Thibaud, J. L., Escriou, C.,    Tissier, R., Retortillo, J. L. and Pouchelon, J. L. (2004) Tissue    Doppler assessment of diastolic and systolic alterations of radial    and longitudinal left ventricular motions in Golden Retrievers    during the preclinical phase of cardiomyopathy associated with    muscular dystrophy. American journal of veterinary research, 65,    1335-1341.-   Chetboul, V., Escriou, C., Tessier, D., Richard, V., Pouchelon, J.    L., Thibault, H., Lallemand, F., Thuillez, C., Blot, S. and    Derumeaux, G. (2004) Tissue Doppler imaging detects early    asymptomatic myocardial abnormalities in a dog model of Duchenne's    cardiomyopathy. European heart journal, 25, 1934-1939.-   Schatzberg, S. J., Anderson, L. V., Wilton, S. D., Kornegay, J. N.,    Mann, C. J., Solomon, G. G. and Sharp, N. J. (1998) Alternative    dystrophin gene transcripts in golden retriever muscular dystrophy.    Muscle & nerve, 21, 991-998.-   Cooper, B. J., Gallagher, E. A., Smith, C. A., Valentine, B. A. and    Winand, N. J. (1990) Mosaic expression of dystrophin in carriers of    canine X-linked muscular dystrophy. Laboratory investigation; a    journal of technical methods and pathology, 62, 171-178.-   Banks, G. B., Gregorevic, P., Allen, J. M., Finn, E. E. and    Chamberlain, J. S. (2007) Functional capacity of dystrophins    carrying deletions in the N-terminal actin-binding domain. Hum Mol    Genet, 16, 2105-2113.-   Beggs, A. H., Hoffman, E. P., Snyder, J. R., Arahata, K., Specht,    L., Shapiro, F., Angelini, C., Sugita, H. and Kunkel, L. M. (1991)    Exploring the molecular basis for variability among patients with    Becker muscular dystrophy: dystrophin gene and protein studies. Am J    Hum Genet, 49, 54-67.-   Chelly, J., Gilgenkrantz, H., Lambert, M., Hamard, G., Chafey, P.,    Recan, D., Katz, P., de la Chapelle, A., Koenig, M., Ginjaar, I. B.    et al. (1990) Effect of dystrophin gene deletions on mRNA levels and    processing in Duchenne and Becker muscular dystrophies. Cell, 63,    1239-1248.-   Le, T. T., Nguyen, T. M., Love, D. R., Helliwell, T. R.,    Davies, K. E. and Morris, G. E. (1993) Monoclonal antibodies against    the muscle-specific N-terminus of dystrophin: characterization of    dystrophin in a muscular dystrophy patient with a frameshift    deletion of exons 3-7. Am J Hum Genet, 53, 131-139.-   Matsumura, K., Burghes, A. H., Mora, M., Tome, F. M., Morandi, L.,    Cornello, F., Leturcq, F., Jeanpierre, M., Kaplan, J. C.,    Reinert, P. et al. (1994) Immunohistochemical analysis of    dystrophin-associated proteins in Becker/Duchenne muscular dystrophy    with huge in-frame deletions in the NH2-terminal and rod domains of    dystrophin. The Journal of clinical investigation, 93, 99-105.-   Muntoni, F., Gobbi, P., Sewry, C., Sherratt, T., Taylor, J.,    Sandhu, S. K., Abbs, S., Roberts, R., Hodgson, S. V., Bobrow, M. et    al. (1994) Deletions in the 5′ region of dystrophin and resulting    phenotypes. Journal of medical genetics, 31, 843-847.-   Novakovic, I., Bojic, D., Todorovic, S., Apostolski, S., Lukovic,    L., Stefanovic, D. and Milasin, J. (2005) Proximal dystrophin gene    deletions and protein alterations in becker muscular dystrophy.    Annals of the New York Academy of Sciences, 1048, 406-410.-   Prior, T. W., Papp, A. C., Snyder, P. J., Burghes, A. H., Bartolo,    C., Sedra, M. S., Western, L. M. and Mendell, J. R. (1993) A    missense mutation in the dystrophin gene in a Duchenne muscular    dystrophy patient. Nature genetics, 4, 357-360.-   Takeshima, Y., Nishio, H., Narita, N., Wada, H., Ishikawa, Y.,    Ishikawa, Y., Minami, R., Nakamura, H. and Matsuo, M. (1994)    Amino-terminal deletion of 53% of dystrophin results in an    intermediate Duchenne-Becker muscular dystrophy phenotype.    Neurology, 44, 1648-1651.-   Winnard, A. V., Mendell, J. R., Prior, T. W., Florence, J. and    Burghes, A. H. (1995) Frameshift deletions of exons 3-7 and    revertant fibers in Duchenne muscular dystrophy: mechanisms of    dystrophin production. Am J Hum Genet, 56, 158-166.-   Warner, L. E., DelloRusso, C., Crawford, R. W., Rybakova, I. N.,    Patel, J. R., Ervasti, J. M. and Chamberlain, J. S. (2002)    Expression of Dp260 in muscle tethers the actin cytoskeleton to the    dystrophin-glycoprotein complex and partially prevents dystrophy.    Hum Mol Genet, 11, 1095-1105.-   Rybakova, I. N., Humston, J. L., Sonnemann, K. J. and    Ervasti, J. M. (2006) Dystrophin and utrophin bind actin through    distinct modes of contact. The Journal of biological chemistry, 281,    9996-10001.

1. A transgenic livestock animal or cell comprising a genomic knockoutof a gene in an artiodactyl that provides for maintenance and/ordevelopment of hair or hair growth.
 2. The livestock animal or cell ofclaim 1 being sheep, goat, or cattle.
 3. The livestock animal or cell ofclaim 1 being pig, miniature pig, or Ossabaw pig.
 4. The livestockanimal or cell of claim 1 wherein the gene is a Hairless gene (HR). 5.The livestock animal or cell of claim 4 wherein exon 2 of HR is targetedfor the knockout of HR.
 6. The livestock animal or cell of claim 1wherein the gene provides for maintenance and/or development of hairgrowth.
 7. The livestock animal or cell of claim 1 wherein the geneprovides for maintenance and/or development of hair.
 8. The livestockanimal of claim 1 having skin denuded of hair.
 9. A method of making atransgenic livestock animal or cell knocking out a gene in anartiodactyl that provides for maintenance and/or development of hair orhair growth.
 10. The method of claim 9 comprising using recombinantAdeno-associated virus (rAAV) homologous recombination to knock out thegene.
 11. The method of claim 9 wherein the gene is a Hairless gene(HR).
 12. The method of claim 11 wherein exon 2 of HR is targeted forthe knockout of HR.
 13. The method of claim 9 comprising performingnuclear transfer of the cell to make the animal.
 14. Tissue isolatedfrom the transgenic livestock of claim 1.